Identification of calibration deviations of pH-measuring devices

ABSTRACT

The invention relates to a comparison unit ( 130 ) configured for determining if a first pH measuring device of a first tank ( 104; 106 ) is affected by a pH-measuring problem, the comparison unit being configured for:
         receiving a first CO2 concentration and a first pH value, the first CO2 concentration being a CO2 concentration of a first gas volume above a medium in a first tank, the first CO2 concentration and the first pH value being measured at a first time when the medium in the first tank is in pH-CO2 equilibrium state with the first gas volume and before said equilibrium state is modified by the metabolism of a cell culture in the first tank, the first pH value being a measured value provided by a first pH measuring device operatively coupled to the first tank ( 102 );   receiving a second CO2 concentration and a second pH value, the second CO2 concentration being a CO2 concentration of a second gas volume above a medium in a second tank, the second CO2 concentration and the second pH value being measured at a second time when the medium in the second tank is in pH-CO2 equilibrium state with the second gas volume and before said equilibrium state is modified by the metabolism of a cell culture, the second pH value being a measured value provided by a second pH measuring device;   comparing the first and second pH values and CO2 concentrations for determining if comparing ( 206 ), by the comparison unit, the first and second pH values and comparing the first and second CO2 concentrations for determining if the first pH measuring device is affected by the pH-measuring problem.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 15/765,222(published as US20180216059A1), filed Mar. 30, 2018, which applicationis a U.S. National Stage entry of International Application No.PCT/EP2016/076173, filed Oct. 28, 2016, which application claims thebenefit of European Patent Application No. 15192389.3, filed Oct. 30,2015, each of which is herein incorporated by reference in its entiretyfor all purposes.

FIELD OF THE INVENTION

The present invention relates to the field of using and calibrating pHmeasuring devices, and more particular to the identification ofcalibration errors and/or pH offset effects resulting from a samplingprocess.

BACKGROUND AND RELATED ART

Using accurately calibrated pH measuring devices is useful or criticalin a great many situations, including chemical or biological laboratorywork, for operating bioreactors, for monitoring harvest reactors, etc.

Bioreactors are commonly used for carrying out chemical processes, inparticular processes performed by living organisms, in a controlledmanner, e.g. in order to obtain a chemical compound, e.g. a particularpeptide, protein, or other kind of chemical substance. A common goal isto operate the bioreactor in a way that the microorganisms or cells areable to perform their desired function with limited production ofimpurities and/or in a time- and cost-efficient manner. Theenvironmental conditions inside the bioreactor, such as temperature,nutrient concentrations, pH, and dissolved gases, among otherparameters, are typically chosen such that the growth and productivityof the organisms is optimized.

In order to determine if the state of a bioreactor and/or the state of acell culture in a bioreactor is at a desired state, e.g. a statecorresponding to a state of a reference bioreactor at a given moment intime when performing a cell culture project, the pH value is repeatedlymeasured. In case the pH value is outside a desired pH-value range,various parameters of the bioreactors (such as feed rate, aeration rate,temperature, stirring rate or the like) may be adapted to change thestate of the bioreactor in a way that the measured pH value in themedium lies within the desired pH value range.

Various control parameters of a bioreactor may be set in dependence on acurrently measured pH value in the medium of a bioreactor. The correctsetting of said parameters determines if a particular cell culture willbe cultivated under very similar/approximately identical conditions as areference culture in another bioreactor. Therefore, in order tosynchronize the state of a bioreactor with the state of another(reference) bioreactor, it is of crucial importance that the pHmeasuring devices of the two compared bioreactors output the same pHvalue for media having the same pH value.

Many other use case scenarios exist where the determination of thecorrect pH value is of crucial importance. For example, at the end of acell culture project, the cell culture and/or its products can be storedin a harvest tank before they are further processed to extract thedesired cell culture products. The harvest tanks need to be closelymonitored to prevent or at least immediately identify any infection orother modification of the conditions within the harvest tank that couldresult in a degradation of its contents. Therefore, the pH value inharvest tanks is repeatedly measured by taking samples and measuring thepH value of the samples.

Often, tank-external pH measuring devices are used for measuring the pHvalue of a sample of the medium contained in a tank, e.g. a bioreactoror harvest tank. However, the process of withdrawing medium samplesbears the risk of an infection of the tank. Moreover, the pH valuemeasured in a sample may deviate from the actual pH value of the mediumwithin the tank due to so called “offset effects”. Offset effects may becaused, for example, during the process of withdrawing the sample andtransporting it to the pH measuring device as the temperature of thesample, the environmental pressure or environmental air composition maydiffer from the respective parameters within the tank. This may resultin a measured pH value in the sample that differs—due to offseteffects—significantly from the actual pH value of the medium in thetank. As a consequent, the pH value measured in the sample does notaccurately reflect the current state in the tank.

Wrongly calibrated pH measuring devices are a further potential sourceof error: typically, pH measuring devices are calibrated withcommercially available reference solutions having a defined pH value.This approach typically requires the withdrawal and re-introduction ofthe pH measuring device from the tank. After re-introduction of the pHmeasuring device, the tank and the pH measuring device contained thereinneed to be autoclaved. This process may have an effect on the alreadycalibrated pH measuring device, resulting in a pH measuring device inthe tank that might indicate a different pH value for the referencesolution than it did before autoclavation. Even in case the pH measuringdevice was not affected by the autoclavation process, there is noguarantee that the pH measuring device was unaffected by theautoclavation. As a result, the measured pH values may be not beconsidered as trustworthy and a re-calibration of the tank-internal pHmeter may be performed to calibrate the tank-internal pH meter by usinga tank-external reference pH meter that measures the pH in a sample ofthe medium of the tank. However, due to offset effects during thesampling process, this will also not guarantee that the tank-internal pHmeter is calibrated correctly.

Regularly taking samples for performing offline pH measurements isburdensome and increases the risk of infecting the bioreactor withundesired germs.

SUMMARY

It is an objective of the present invention to provide for an improvedsystem and method for determining if a pH measuring device is affectedby a pH-measuring problem and for re-calibrating a pH meter as specifiedin the independent claims. Embodiments of the invention are given in thedependent claims. Embodiments of the present invention can be freelycombined with each other if they are not mutually exclusive. Embodimentsof the invention may take advantage of the ease with which a CO2concentration in the air volume in a tank can be measured for improvingand facilitating the identification of pH measuring problems andcalibration problems. According to one beneficial aspect, CO2concentrations measured in two or more different tanks may be used foridentifying pH-measuring deviations of the pH measuring devices whichare operatively coupled to the different tanks.

In one aspect, the invention relates to a method for determining if afirst pH measuring device operatively coupled to a first tank isaffected by a pH-measuring problem. The pH measuring problem is that thefirst pH measuring device is calibrated differently than a second pHmeasuring device operatively coupled to a second tank. For example, thissituation can be problematic if the second tank is a referencebioreactor and the first tank is another bioreactor whose state shall becompared and synchronized with the state of the reference bioreactor.The method comprises:

-   -   receiving by a comparison unit, a first CO2 concentration and a        first pH value, the first CO2 concentration being a CO2        concentration of a first gas volume above a medium (M1) in the        first tank, the first CO2 concentration and the first pH value        being measured at a first time, the first time being a time when        the medium in the first tank is in pH-CO2 equilibrium state with        the first gas volume at a predefined temperature and pressure,        said equilibrium state being unaffected by the metabolism of any        cell culture, the first pH value being a measured value provided        by the first pH measuring device (108; 146);    -   receiving, by the comparison unit, a second CO2 concentration        and a second pH value, the second CO2 concentration being a CO2        concentration of a second gas volume above the same type of        medium (M1) contained in the second tank, the second CO2        concentration and the second pH value being measured at a second        time, the second time being a time when the medium in the second        tank is in pH-CO2 equilibrium state with the second gas volume        at the predefined temperature and pressure, said equilibrium        state being unaffected by the metabolism of any cell culture,        the second pH value being a measured value provided by the        second pH measuring device;    -   comparing, by the comparison unit, the first and second pH        values and comparing the first and second CO2 concentrations for        determining if the first pH measuring device is affected by the        pH-measuring problem.

According to embodiments, the determination that the first pH measuringdevice has a pH measuring problem is made in case:

-   -   first and second CO2 concentrations are identical and the first        and second pH values differ from each other by more than a        threshold value; or    -   the first and second pH values are identical and the first and        second CO2 concentrations differ from each other by more than a        further threshold value; or    -   a first data value differs from a second data value by more than        a further threshold, the first data value being derived from the        first pH value and the first CO2 concentration, the second data        value being derived from the second pH value and the second CO2        concentration.

The first tank can be, for example, a bioreactor or a harvest tank. Thesecond tank can be, for example a bioreactor, in particular a referencebioreactor, or a harvest tank, in particular a reference harvest tank.

Embodiments of the invention may be advantageous as they allow to moreaccurately compare the pH values of the media in two or more tanks, e.g.a reference tank and one or more monitored or controlled tanks. Thereference pH profile of the reference tank may be obtained several days,weeks or years prior to obtaining the measurement values in the tank(s)to be monitored. Alternatively, the first and second tanks may beoperated in a basically concurrent manner. In a further beneficialaspect, offline measurements of the pH can be omitted, thereby avoidinga contamination of the tanks and avoiding an inaccurate comparison of pHvalues as sampling effects may create a significant variability of themeasured pH value.

According to a further beneficial aspect, CO2 concentrations measured ina particular tank may be used for computing an absolute, expected pHvalue and may allow identifying any deviations of the actually measuredpH value from the expected pH value. Said deviation may be an indicatorfor a pH measuring problem, e.g. a calibration problem.

In a further aspect, the invention relates to a method for determiningif a first pH measuring device operatively coupled to a first tank isaffected by a pH-measuring problem. The problem is that the first pHmeasuring device is calibrated wrongly (and thus does not only produce apH value that is wrong relative to a pH value provided by a reference pHmeasuring device, but that produces a pH value that is wrong in absoluteterms). The method comprises:

-   -   receiving, by a comparison unit, a first CO2 concentration and a        first pH value, the first CO2 concentration being a CO2        concentration of a first gas volume above a medium (M1) in the        first tank, the first CO2 concentration and the first pH value        being measured at a first time, the first time being a time when        the medium in the first tank is in pH-CO2 equilibrium state with        the first gas volume at a predefined temperature and pressure,        said equilibrium state being unaffected by the metabolism of any        cell culture, the first pH value being a measured value provided        by the first pH measuring device;    -   computing, by the comparison unit, a second pH value as a        function of the first CO2 concentration, the second pH value        being the pH value predicted for said type of medium (M1) when        said medium is in pH-CO2 equilibrium state with a second gas        volume above said medium (M1) at the predefined temperature and        pressure, the second gas volume in said equilibrium having a        second CO2 concentration that is identical to the first CO2        concentration, said equilibrium state being unaffected by the        metabolism of any cell culture;    -   comparing, by the comparison unit, the first and second pH        values for determining if the first pH measuring device is        affected by the pH-measuring problem.

This may be advantageous as this method allows to determine, providedsome properties of the medium are known, the correct, absolute pH valueof the medium using the CO2 concentration in the air volume of the tankthat may be measured in the offgas as input. By comparing the computed,accurate pH value derived from the CO2 concentration with the actuallymeasured pH value, it is possible to detect calibration errors.

According to embodiments, the comparison unit outputs a warning signalthat in case it was determined that the first pH measuring device isaffected by the pH measuring problem. This may allow an operator to takeappropriate action, e.g. re-calibrate the pH measuring device, replacethe pH measuring device, etc.

Computing an Expected pH Value Using the Offgas CO2 Concentration

According to some examples related to the use of “minimalistic” media,e.g. salt solutions being basically free of substances acting as abuffer, the calculation of a pH value based on carbon dioxide in the gasphase in carbonate buffered systems can be performed as follows:

The concentration of carbon dioxide that is dissolved in a liquid isproportional to carbon dioxide partial pressure (pCO2) in the gas phaseand can be calculated using respective proportional factors.Proportional factors depend on the liquid and temperature as well aspressure.

For example, the solubility coefficient for CO2 in various liquids isknown and can be derived from literature or can be determinedexperimentally. The solubility coefficient in blood at 37 degC. is about0.0304 mmol×L−1×mmHg−1 [Löffler, G., Petrides, P. E., PhysiologischeChemie, Springer-Verlag, volume 4, 1990, p. 24]. According to Sazonovand Shaw, the Bunsen coefficient is defined as the volume of saturatinggas reduced 273.15 and 1 bar, which is absorbed by unit volume of puresolvent at the temperature of measurement and partial pressure of 1 bar.Dimension is therefore volume/volume or dimensionless. This coefficientis based on Henry's Law, claiming that in equilibrium the partialpressure of a gas is directly proportional to the concentration of thisgas that is solved in a correlated solution. Solubility coefficientsdepend on the respective solution [Gros, J. B., Dussap, C. G., Catté,M., Estimation of O2 and CO2 solubility in microbial culture media.Biotechnology Progress 1999, 15, 923-927].

If the solubility coefficient of a medium is known, concentration ofdissolved carbon dioxide can be calculated by carbon dioxideconcentrations in the gas phase. Concentrations in the gas phase, e.g.in bioreactors, can be directly measured using an off gas analyzer.

Carbon Dioxide and Carbonic Acid-Base Equilibria

CO_(2(g))→CO_(2(aq))  Equation 1

Dissolved CO_(2(aq)) is hydrated in water (H₂O) to carbonic acid(H₂CO₃).CO_(2(aq))+H₂O⇄H₂CO₃  Equation 2

In aqueous solutions at neutral pH and 37 degC hydrated species carbonicacid is almost nonexistent; both species therefore can be combined toH2CO3*(CO2(aq)+H2CO3=H2CO3). At typical fermentation pH around 7.00,H₂CO₃* dissociates to bicarbonate (HCO3−) and a proton (H+). Carbonate(CO32−), the deprotonated species is almost nonexistent at neutral pHvalues.

Aqueous CO₂ (aq) can dissolve limestoneCaCO₃+CO₂(aq)+H₂O⇄Ca²⁺(aq)+2HCO₃ ⁻(aq)and can react with the water to form carbonic acidCO₂(aq)+H₂O⇄H₂CO₃(aq)

Only a small fraction exists as the acid, so the dissociation constant Kis

$K = {\frac{\lbrack {H_{2}{{CO}_{3}({aq})}} \rbrack}{\lbrack {{CO}_{2}({aq})} \rbrack}.}$

Carbon Dioxide and Carbonic Acid-Base Equilibria

Dissolved CO2 in the form of H2CO3 may loose up to two protons throughthe acid equilibriaH₂CO*₃⇄HCO₃ ⁻+H⁺ K_(S1)=10^(−6.35)  Equation 3HCO₃ ⁻⇄CO₃ ²⁻+H⁺ K_(S2)=10^(−10.33)  Equation 4

Dissociation constants K_(S1) and K_(S2), are given here for standardconditions (298, 15 K, ion strength I_(c)=0 M) [Goudar, C. T. C.,Matanguihan, R., Long, E., Cruz, C., et al., Decreased pCO2 accumulationby eliminating bicarbonate addition to high cell-density cultures.Biotechnology and Bioengineering 2007, 96, 1107-1117].

For cell culture conditions temperatures of 37 degC as well as ionstrengths of 0.1 M can be used. This will change the respectivedissociation constants to 10⁻⁶⁰⁷ and 10^(−10.04), respectively.

Ratio of all species carbon dioxide (H₂CO₃*), bicarbonate HCO₃ ⁻ andcarbonate CO₃ ²⁻ can be calculated using theHenderson-Hasselbalch-Equation.

Equilibrium equations:

$\begin{matrix}{K_{S1} = \frac{\lbrack H^{+} \rbrack\lbrack {HCO}_{3}^{-} \rbrack}{\lbrack {H_{2}{CO}_{3}} \rbrack}} & {{Equation}5}\end{matrix}$ $\begin{matrix}{K_{S2} = \frac{\lbrack H^{+} \rbrack\lbrack {CO}_{3}^{2 -} \rbrack}{\lbrack {H_{2}{CO}_{3}^{-}} \rbrack}} & {{Equation}6}\end{matrix}$

Acid equilibrium equations can be solved to give the fraction ∝ ofrespective carbonates as a function of proton concentration, hence pH:

$\begin{matrix}{\propto_{H_{2}{CO}_{3}}{= \frac{\lbrack H^{+} \rbrack^{2}}{\lbrack H^{+} \rbrack^{2} + {\lbrack H^{+} \rbrack*K_{S1}} + {K_{S1}K_{S2}}}}} & {{Equation}7}\end{matrix}$ $\begin{matrix}{\propto_{{HCO}_{3}^{-}}{= \frac{\lbrack H^{+} \rbrack*K_{S1}}{\lbrack H^{+} \rbrack^{2} + {\lbrack H^{+} \rbrack*K_{S1}} + {K_{S1}K_{S2}}}}} & {{Equation}8}\end{matrix}$ $\begin{matrix}{\propto_{{HCO}_{3}^{-}}{= \frac{K_{S1}K_{S2}}{\lbrack H^{+} \rbrack^{2} + {\lbrack H^{+} \rbrack*K_{S1}} + {K_{S1}K_{S2}}}}} & {{Equation}9}\end{matrix}$

Relative H2CO3 concentration is in effect CO2 (aq) in equilibrium withwater.

Computing the pH

In the following, an exemplary computation of an expected pH based oncarbon dioxide concentration in water at 37 degrees Celsius will bedescribed:

If carbon dioxide concentration in gas phase is known, dissolved carbondioxide concentration can be calculated by the use of the Bunsencoefficient, valid at atmospheric pressure at 37 degC [Löffler, G.,Petrides, P. E., Physiologische Chemie, Springer-Verlag, volume 4, 1990,p. 24].CO_(2aq)=0.0304 [mmol/L*mmHg]*pressure*carbon dioxide concentrationgaseousphase/100.

The pressure in this case is atmospheric pressure of 750.06 mmHg.

There may be 10% carbon dioxide in the gas phase of a tank. The carbondioxide is given in [%]. The partial pressure of CO₂ in mmol/L (CO_(2áq)[mmol/L]) is in this case computed according to 750.06mmHg*10%/100=75,006 mmHg. We have then mmol/L CO_(2áq).

Then, the proton concentration of the first equation H2CO3→H++HCO3− iscomputed via equation 5 and equation 6:

H⁺ concentration equation 5=1,01468E−06 mmol/L.

H⁺ concentration equation 6=1,06941E−10 mmol/L.

The overall H⁺ concentration therefore is 1,01468E−06 mmol/L+1,06941E-10mmol/L=1,01479E−06 mmol/L.

The contribution of equation 5 to the overall H⁺ concentration is largerthan that of equation 6.

pH is then computed according to pH=−log(1,01479E−06)=5,99.

The CO2 concentration in the gas phase can be computed based onbicarbonate concentration and pH according to:

${{CO}_{2}\lbrack\%\rbrack} = {\frac{\{ \frac{( \frac{\lbrack {10^{{- p}H}} \rbrack^{2}}{\lbrack {10^{{- p}H}} \rbrack^{2} + {\lbrack {10^{{- p}H}} \rbrack*K_{S1}} + {K_{S1}K_{S2}}} )*C}{b} \}}{750.06{mm}{Hg}}*100}$

With b being the Bunsen coefficient of 0.0304 mmol/L*mmHg at normalatmospheric pressure of 750.06 mmHg, pH being the pH value of solution,and C being the concentration of bicarbonate in [mmol/L].

First, the amount of CO2 dissolved in water (aq) under normal atmosphereof pressure is computed using Henry's Law[CO2(aq)]=K_(CO2)*H,  Equation 10wherein K_(CO2) is the CO2 concentration measured in the gasphase/offgas and H is the Henry solubility.

After having determined [CO2(aq)] (equation 10) and K_(s1) (equation 5),the formula

$\begin{matrix}{K_{S1} = \frac{\lbrack H^{+} \rbrack^{2}}{\lbrack {{{CO}2}({aq})} \rbrack}} & {{Equation}11}\end{matrix}$

Can be resolved for computing the pH aspH=log₁₀([H⁺])=log₁₀(√{square root over (K_(S1)[CO2(aq)])}).Computing the Expected pH Value Using a Medium Specific Relation

According to embodiments, the comparison unit, for computing of thesecond pH value, reads a medium-specific relation from a data storagemedium. The medium-specific relation is specific for the medium M1 ofthe first tank and indicates a relation between the pH value of themedium M1 and a respective fraction of CO2 gas in a gas volume when saidmedium is in pH-CO2 equilibrium state with said gas volume and lacks acell culture. The comparison unit inputs the first CO2 concentrationinto the medium specific relation for calculating an absolute pH valueexpected for the medium in pH-CO2 equilibrium at the predefinedtemperature and pressure and under the absence of a cell culture. Theabsolute pH value is used as the computed second pH value.

This may be advantageous as a medium specific relation that correlatesthe pH value in the medium with a CO2 concentration in the offgas inCO2-pH equilibrium state can be obtained empirically for any kind ofmedium, including media comprising a plurality of substances which havean impact on the pH-CO2 equilibrium.

According to embodiments, the medium-specific relation is an equationPPH_(M1)(CO2)=REL-M1 (CO2) obtained by mathematically fitting multipleempirically determined pairs of a pH-value of the medium (M1) and arespectively measured fraction of CO2 gas in a gas volume. PPH_(M1)(CO2)is the predicted pH value in a medium (M1) when said medium lacks a cellculture and is at pH-CO2 equilibrium with a gas volume above saidmedium, said gas volume comprising the CO2 concentration used as inputparameter. The CO2 is an input parameter value and represents the CO2concentration in a gas volume above the medium (M1) in pH-CO2equilibrium state under the absence of the cell culture. REL-M1 is a setof one or more parameters (a1, a2, b1, b2, b3) connected by operators.The parameters have been obtained empirically by a method comprising:

-   -   adjusting samples of the medium (M1) lacking the cell culture to        multiple different pH values, thereby letting the samples reach        pH-CO2 equilibrium with the gas volume above the medium in the        respective sample,    -   determining the fraction of CO2 gas in a in respective gas        volume being in ph-CO2 equilibrium with the medium in the        samples,    -   plotting the determined CO2 gas fractions against the respective        equilibrium pH values of the samples,    -   fitting a curve in the plotted values and deriving the        parameters (a1, a2 or b1, b2, b3) of the medium-specific        relation from the fitted curve.

For example, the medium specific relation can be determined in a specialcontainer or bioreactor that comprises a CO2 offgas sensor and an onlinepH measuring device. The conditions used for determining the mediumspecific relation, e.g. temperature and pressure, are preferentially thesame or very similar to the temperature and pressure prevailing whenmeasuring the first pH value.

According to embodiments, the medium M1 in the tank whose pH value ismeasured is a medium of defined composition that is known to have thesame composition like the medium used for empirically generating themedium-specific relation. For example, the medium in the tank and themedium used for generating the medium specific relation may be preparedby the operator of the tank or may be retrieved from a provider whodiscloses all components of the medium and the respectiveconcentrations. Thus, the expressions “same type of medium” or the “samemedium” as used herein both refer to media having the same compositionat least in respect to all components having an impact on the pH-CO2equilibrium.

For example, the medium may be a bicarbonate buffer that is free of anyother substances (except the bicarbonate) which have an impact on thepH-CO2 equilibrium. Some providers of commercially available media donot disclose the complete list of ingredients. By preparing the bufferhaving a defined, limited set of components, the operator of a tank mayensure that the medium in the tank has exactly the same composition likethe medium used for generating the medium specific relation. This mayprevent an erroneous detection of a pH measuring problem or could themissing of a real pH measuring problem.

According to embodiments, the determination that the first pH measuringdevice has a pH measuring problem is made in case the first and secondpH values differ from each other by more than a threshold value.Likewise, the determination that the first pH measuring device has a pHmeasuring problem is made in case a first data value differs from asecond data value by more than a further threshold, the first data valuebeing derived from the first pH value, the second data value beingderived from the second pH value. For example, the threshold may be setby an operator of the tank to which the first pH measuring device iscoupled and may depend on the accuracy requirements of the operator orthe project for which the tank and the medium is used.

According to embodiments, the first tank is a bioreactor or a harvesttank or a calibration box.

According to a further beneficial aspect, CO2 concentrations measured ina particular tank may be used for calibrating or re-calibrating anerroneously calibrated pH measuring device.

In a further aspect, the invention relates to a method for calibratingor re-calibrating a first pH measuring device. The method comprisescomparing pH values and CO2 concentrations measured in the first and inthe second tank as described above for embodiments of the invention fordetermining that the first pH measuring device is affected by apH-measuring problem; and calibrating the first pH measuring device suchthat it outputs the same pH value like the second pH measuring device incase the first and second CO2 concentrations are identical.Alternatively, the method comprises computing an expected second pHvalue from a first CO2 concentration measured in the offgas of a firsttank as described above for embodiments of the invention for determiningthat the first pH measuring device in the first tank is affected by apH-measuring problem; and calibrating the first pH measuring device suchthat it outputs the same pH value like the pH value computed as afunction of the first CO2 concentration.

According to a further beneficial aspect, CO2 concentrations measured ina particular tank may be used for calibrating or re-calibrating anerroneously calibrated pH measuring device without removing andre-inserting the pH measuring device from and to the tank.

In a further aspect, the invention relates to a method of operating atank comprising a first pH measuring device. The first pH measuringdevice is an online measuring device, the method comprising:

-   -   growing a cell culture in the tank, the tank comprising a growth        medium, thereby repeatedly measuring the pH in the growth medium        by the first pH measuring device;    -   replacing the growth medium and the cell culture contained        therein in the tank with a medium (M1) for which a relation        between pH and CO2 in equilibrium is known; for example, said        medium M1 may be a medium whose corresponding medium-specific        relation is stored in a data storage medium accessible to a        comparison unit that performs the determination if a pH        measuring problem exists, or the medium M1 may be a solely        bicarbonate-buffered medium allowing the computation of the        absolute pH value from the CO2 offgas concentration at pH-CO2        equilibrium;    -   after having replaced the growth medium, computing an expected        second pH value from the measured CO2 off gas concentration as        described above for embodiments of the invention for determining        that the first pH measuring device is affected by a pH-measuring        problem;    -   if a pH measuring problem was detected, calibrating the first pH        measuring device such that it outputs the same pH value like the        pH value computed as a function of the first CO2 concentration        for the medium (M1);    -   after having calibrated the first pH measuring device, replacing        the medium in the tank with the growth medium.

Said features may be advantageous as it is not necessary any more towithdraw the pH measuring device from the tank, execute some calibrationtests, optionally recalibrate it, autoclave the pH meter and reintroducethe autoclaved pH meter into the tank. Rather, the pH measuring device,which may be an online pH measuring device located within the tank, canbe checked and re-calibrated in the tank. This may reduce thecontamination risk and save time.

According to a further beneficial aspect, CO2 concentrations measured ina particular tank may be used for identifying pH offset effects causedby taking a sample of the medium in the tank.

In a further aspect, the invention relates to a method of determining pHoffset effects caused by taking a medium sample from a first tank. Themethod comprising providing a tank-external, offline pH measuring device(160) and providing the first tank. The first tank comprises a first pHmeasuring device. The first pH measuring device is an online pHmeasuring device located within the first tank and is at least partiallysurrounded by the medium (M1) in the first tank. The method furthercomprises:

-   -   calibrating the first (tank-internal) pH measuring device by        computing a predicted, absolute pH value from a measured CO2        offgas concentration of the first tank for determining if the        first (tank-internal) pH measuring device is affected by a        pH-measuring problem; and calibrating (if a pH measuring problem        was detected) the first (tank-internal) pH measuring device such        that it outputs the same pH value like the pH value computed as        a function of the first CO2 concentration;    -   transferring the tank-external, offline pH measuring device into        a calibration box comprising the same type of medium (M1) as the        first tank; and calibrating (if a pH measuring problem was        detected) the tank-external, offline pH measuring device by        computing a predicted, absolute pH value from a measured CO2        offgas concentration of the calibration box for determining if        the tank-external, offline pH measuring device is affected by a        pH-measuring problem; and calibrating (if a pH measuring problem        was detected) the tank-external, offline pH measuring device        such that it outputs the same pH value like the pH value        computed as a function of the CO2 concentration measured in the        off gas of the calibration box; thus, the calibration box is        used as the tank comprising the pH measuring device to be        calibrated and is used as a container whose CO2 offgas sensor is        used for measuring the CO2 concentration used as input for        computing the second pH value to be used for calibrating the        tank-external, offline pH measuring device.

After having calibrated the first pH measuring device and thetank-external pH measuring device, the method comprises:

-   -   measuring, by the first pH measuring device, a first current pH        value of the medium in the first tank, the first current pH        value being an online-measurement value;    -   taking a sample of the medium of the first tank and filling the        sample into a portable container (162);    -   positioning the tank-external pH measuring device such that it        is at least partially surrounded with the medium in the sample        container;    -   measuring, by the tank-external pH measuring device, a second        current pH value of the medium in the sample container, the        second current pH value being an offline-measurement value;    -   in case the first and the second current pH values differ by        more than a threshold, determining that the sampling process        caused an pH offset effect.

According to embodiments, the method further comprises:

-   -   receiving a third CO2 concentration and a third pH value, the        third CO2 concentration being a CO2 concentration of a third gas        volume above the medium in the first tank, the third CO2        concentration and the third pH value being measured at a third        time, the third time being a time when the medium in the first        tank is in pH-CO2 equilibrium state at a predefined temperature        and pressure with the third gas volume and after said        equilibrium state is modified by the metabolism of the cell        culture in the first tank, the third pH value being a measured        value provided by the first pH measuring device;    -   receiving a fourth CO2 concentration and a fourth pH value, the        fourth CO2 concentration being a CO2 concentration of a fourth        gas volume above the medium in the second tank, the fourth CO2        concentration and the fourth pH value being measured at a fourth        time, the fourth time being a time when the medium in the second        tank is in pH-CO2 equilibrium state at the predefined        temperature and pressure with the second gas volume and after        said equilibrium state is modified by the metabolism of the cell        culture in the second tank, the fourth pH value being a measured        value provided by the second pH measuring device, the lapsed        time between the third time and the inoculation of the first        tank being identical to the lapsed time between the fourth time        and the inoculation of the second tank;    -   receiving a measured first oxygen uptake rate of the cell        culture in the first tank at the third time;    -   receiving a measured second oxygen uptake rate of the cell        culture in the second tank at the fourth time;    -   in case the first and second oxygen uptake rates are identical,        comparing the third and fourth pH values and CO2 concentrations        for determining if the first and second pH measuring devices are        calibrated differently.

For example, said steps may be performed by a comparison unit, e.g. apiece of program logic that monitors and/or controls the pH measurementsand calibration states of one or more pH measuring devices. In addition,the comparison unit or a control unit coupled to the comparison unit maymonitor and/or control the state of one or more tanks.

Accurately measuring and minimizing offset effects of a sampling processmay be highly advantageous as the pH value measured in a sample mayoften be used as an important control parameter of bioreactors. The pHvalue measured in a sample of the medium of a bioreactor may often bethe basis for taking corrective actions, e.g. for adding a basicsubstance, increasing or decreasing the temperature or feed rate may beperformed. By calibrating the tank-internal pH measuring device as wellas the tank-external pH measuring device based on a measured CO2concentration, both pH measuring devices can be calibrated with respectto a highly accurate, absolute pH value that can be derived exactly fromthe CO2 concentration. Thus, calibration differences can be minimized.As a consequence, when operating the tank and regularly taking mediasamples, any difference in the pH value of the tank-internal pHmeasuring device and the tank-external pH measuring device can clearlybe ascribed to sampling effects, not to calibration differences. Thus,the effect of the sampling process can be determined more accurately andcan be filtered out (e.g. by computationally adding or subtracting thepH difference caused by the sampling process) from the pH value measuredby the tank-external pH measuring device.

According to embodiments, the first pH measuring device is at leastpartially surrounded by the medium within the first tank. The first tanklacks means for manually or automatically taking a sample of the mediumin the second tank. Alternatively, the first tank comprises means formanually or automatically taking a sample of the medium in the firsttank, but all openings of the sampling means are kept close during atime interval after filling the medium in the first tank and beforeadding a cell culture to the medium in the first tank. This may reducethe risk of contaminating the tank with microbes.

According to embodiments, the second pH measuring device is at leastpartially surrounded by the medium within the second tank. The secondtank lacks means for manually or automatically taking a sample of themedium in the second tank.

Alternatively, the second tank comprises means for manually orautomatically taking a sample of the medium in the second tank, but allopenings of the sampling means are kept close during a time intervalafter filling the medium in the second tank and before adding a cellculture to the medium in the second tank.

According to embodiments, the method for determining pH measuringproblems described herein for embodiments of the invention used fordetermining if the second pH measuring device is calibrated differentlythan the first pH measuring device, the determination if the first andsecond pH measuring device are calibrated differently being performedwhile the second pH measuring device is at least partially surroundedwith the medium in the second tank and without taking a sample of themedium of the second tank for performing said determination.

According to embodiments, comparing the pH values measured by two ormore different pH measuring devices, the determination if the first pHmeasuring device is affected by a pH measuring problem is performed byusing the first and second CO2 concentrations and the first and secondpH values as the only data input for said determination.

According to embodiments, comparing the pH value measured by aparticular pH measuring device with an expected pH value computed fromthe measured CO2 concentration, the determination if the first pHmeasuring device is affected by a pH measuring problem is performed byusing the measured pH value and the measured CO2 concentration of thetank comprising said pH measuring device as the only data input for saiddetermination.

Said features may be advantageous as no offline-measurements may berequired any more for determining pH measuring problems.

According to embodiments, the measuring of the first pH value isperformed as an online-measurement and the first pH measuring device isat least partially surrounded with the medium in the first tank. In thiscase, the first pH measuring device being operatively coupled to thefirst tank is a tank-internal pH measuring device of the first tank.

In addition, or alternatively, the measuring of the second pH value isperformed as an online measurement and the second pH measuring device isat least partially surrounded by the medium in the second tank. In thiscase, the second pH measuring device being operatively coupled to thesecond tank is a tank-internal pH measuring device of the second tank.

According to embodiments, the measuring of the first CO2 concentrationis performed as an online-measurement by a first CO2 sensor in the offgas of the first tank for providing the first CO2 concentration.

According to embodiments, the measuring of the second CO2 concentrationis performed as an online-measurement by a second CO2 sensor in the offgas of the second tank for providing the second CO2 concentration.

According to embodiments, the comparison unit performs, in case ofdetermining that the first pH measuring device is affected by a pHmeasuring problem, one or more of the following steps: outputting awarning message; automatically performing or triggering the performingof a recalibration of the first pH measuring device; or automaticallyperforming or triggering the performing of a replacement of the first pHmeasuring device by a new first pH measuring device.

According to embodiments, the first tank differs from the second tank inrespect to one or more of the following features:

-   -   a) the gas volume in the tank,    -   b) the medium volume in the tank,    -   c) the Reynolds number of the tank,    -   d) the Newton number of the tank,    -   e) the dimensions of the tank,    -   f) geometrical features of the tank and/or tank baffles,    -   g) the stirrer configuration,    -   h) the stirring rate,    -   i) the volumetric mass transfer coefficient for oxygen (kLa) of        the tank,    -   j) total gas influx rate and/or O2 influx rate and/or N2 influx        rate and/or CO2 influx rate,    -   k) power input,    -   l) pressure in the tank,    -   m) gas bubble hold time in the medium,    -   n) gas bubble size and distribution in the medium,    -   o) surface speed,    -   p) a parameter calculated as a derivative from one or more of        the parameters a)-o);    -   q) the geographic location of the two tanks.

In a further aspect, the invention relates to a comparison unitconfigured for:

-   -   receiving, a first CO2 concentration and a first pH value, the        first CO2 concentration being a CO2 concentration of a first gas        volume above a medium (M1) in a first tank, the first CO2        concentration and the first pH value being measured at a first        time, the first time being a time when the medium in the first        tank is in pH-CO2 equilibrium state with the first gas volume at        a predefined temperature and pressure, said equilibrium state        being unaffected by the metabolism of any cell culture, the        first pH value being a measured value provided by the first pH        measuring device;    -   receiving a second CO2 concentration and a second pH value, the        second CO2 concentration being a CO2 concentration of a second        gas volume above the same type of medium (M1) contained in the        second tank, the second CO2 concentration and the second pH        value being measured at a second time, the second time being a        time when the medium in the second tank is in pH-CO2 equilibrium        state with the second gas volume at the predefined temperature        and pressure, said equilibrium state being unaffected by the        metabolism of any cell culture, the second pH value being a        measured value provided by the second pH measuring device;    -   comparing the first and second pH values and comparing the first        and second CO2 concentrations for determining if the first pH        measuring device is affected by the pH-measuring problem.

In a further aspect, the invention relates to a comparison unitconfigured for:

-   -   receiving a first CO2 concentration and a first pH value, the        first CO2 concentration being a CO2 concentration of a first gas        volume above a medium (M1) in a first tank, the first CO2        concentration and the first pH value being measured at a first        time, the first time being a time when the medium in the first        tank is in pH-CO2 equilibrium state with the first gas volume at        a predefined temperature and pressure, said equilibrium state        being unaffected by the metabolism of any cell culture, the        first pH value being a measured value provided by the first pH        measuring device;    -   computing a second pH value as a function of the first CO2        concentration, the second pH value being the pH value predicted        for said type of medium (M1) when said medium is in pH-CO2        equilibrium state with a second gas volume above said medium        (M1) at the predefined temperature and pressure, the second gas        volume in said equilibrium having a second CO2 concentration        that is identical to the first CO2 concentration, said        equilibrium state being unaffected by the metabolism of any cell        culture;    -   comparing the first and second pH values for determining if the        first pH measuring device is affected by the pH-measuring        problem.

In a further aspect, the invention relates to a system configured formonitoring and/or controlling a state of a first tank. The systemcomprises:

-   -   the comparison unit according to embodiments of the invention;    -   a control unit operatively coupled to the comparison unit; and    -   the first tank and the first pH measuring device;    -   the control unit being configured to monitor and/or control a        state of a cell culture in the first tank, thereby using pH        values repeatedly measured by the first pH measuring device as        input.

According to embodiments, the system is configured for using pH valuesof the first and second pH measuring device and the first and second CO2concentrations as an input parameter for monitoring and/or minimizingdeviations of a state of the first tank from the state of the secondtank by analyzing at least said input parameters.

In the following, embodiments and examples will be described by makingreference to bioreactors. However, bioreactors are only one type of tankwhere embodiments of the invention may be applied. Other examples areharvest tanks and calibration boxes.

In one aspect, the invention relates to a method comprising:

-   -   receiving, by a comparison unit, a first CO2 concentration and a        first pH value. The first CO2 concentration is a CO2        concentration of a first gas volume above a medium in the first        bioreactor. The first CO2 concentration and the first pH value        are measured at a first time. The first time is a time when the        medium in the first bioreactor is in pH-CO2 equilibrium state at        a predefined temperature and pressure with the first gas volume        and before said equilibrium state is modified by the metabolism        of a cell culture in the first bioreactor. The first pH value is        a measured value provided by a first pH measuring device        operatively coupled to a first bioreactor;    -   receiving, by the comparison unit, a second CO2 concentration        and a second pH value. The second CO2 concentration is a CO2        concentration of a second gas volume above a medium in the        second bioreactor. The second CO2 concentration and the second        pH value are measured at a second time. The second time is a        time when the medium in the second bioreactor is in pH-CO2        equilibrium state at the predefined temperature and pressure        with the second gas volume and before said equilibrium state is        modified by the metabolism of a cell culture in the second        bioreactor. The second pH value is a measured value provided by        a second pH measuring device operatively coupled to a second        bioreactor. The medium in the first bioreactor is the same as        the medium in the second bioreactor;    -   comparing, by the comparison unit, the first and second pH        values and the second CO2 concentrations for determining if the        first and second pH measuring devices are calibrated differently        or for determining if the first and second pH measuring devices        output different pH values due to offset effects of a sampling        process performed for measuring the first or second pH value in        a sample of the medium of a respective one of the first and        second bioreactors.

For example, the second bioreactor may be used as a reference bioreactorand the first bioreactor may be used as another bioreactor in which acell culture project shall be run basically in the same way as areference cell culture having been cultivated previously in thereference bioreactor. It may also be the case that the first and secondbioreactors shall be run synchronously, whereby the respective pH valuesare repeatedly measured in order to compare the state of thebioreactors. The parameter comparison may be performed, for example, inorder to automatically, semi-automatically or manually initiateappropriate actions in order to prevent a state deviation of said twobioreactors.

It has been observed that calibration errors of the first or second pHmeasuring devices are common error sources resulting in a failure toaccurately compare the states of the two bioreactors and/or toaccurately reproduce a state profile derived from the second bioreactorin the first bioreactor. Calibration errors are common error sourcesboth for offline and online pH measurement approaches.

Further, it has been observed that in case offline pH measurements areperformed in the first and/or second bioreactor, the obtained offline pHvalue may not accurately reflect the current pH value of the medium inthe bioreactor. For example, a medium sample may be regularly drawn froma bioreactor in order to regularly perform an offline pH measurement insaid samples. The sampling process before the pH value can be measuredin the medium sample takes time. In the meantime, the pH value in thebioreactor from which the sample was drawn may have significantlychanged. Further, it may happen that the temperature in the sample dropsduring the sampling process, or the pH value in the sample shifts due toa change in the CO2 concentration in the gas volume above the medium ofthe sample compared to the gas volume above the medium in thebioreactor. All said factors may influence the pH value in the mediumsample and lead to a so called “offset effect”.

An “offset effect” as used herein is a pH value offset by which a pHvalue measured in a medium sample of a bioreactor at a particular timediffers from a pH value that would be measured at said particular timedirectly in the medium of the bioreactor. Thus, calibration errors ofthe first or second pH measuring device and/or offset effects (in casethe first and/or second pH measuring device is an offline measuringdevice) may result in a failure to accurately compare and/or synchronizethe state of two bioreactors based on pH measurement values.

Embodiments of the invention take advantage of the fact that in case twoidentical solutions/media are in pH-CO2 equilibrium state at a giventemperature and pressure and a given pH value, the volume above saidmedia have the same CO2 concentration. Likewise, given a particular CO2concentration in the volumes above said two media in equilibrium state,the pH values of said two media are identical as a consequence of thetwo media being in pH-CO2 equilibrium. As the two media are free of anycells whose metabolism could shift the pH-CO2 equilibrium state, anydeviation of the first and second pH value given identical measured CO2concentrations is used as an indication that the two pH measuringdevices are calibrated differently and/or that the difference is causedby an offset effect of an offline pH measurement.

For example, the “predefined” or “given” temperature of the secondbioreactor can be any temperature and pressure that is suitable forinitiating and/or operating the second bioreactor. For example, thesecond temperature could be 20° C. and the second pressure could benormal atmospheric pressure. The second temperature and pressure may bemeasured and the temperature and pressure of the first bioreactor(referred herein as “first temperature” and “first pressure” can becontrolled and adapted such that the first and second temperatures areidentical or approximately identical and the first and second pressuresare identical or approximately identical.

Embodiments of the invention may be advantageous for multiple reasons:

The comparison of the CO2 concentrations and the pH values at a timebefore the pH value of the medium is affected by any metabolicactivities of the cell culture allows to check if the first pH measuringdevice is calibrated differently than the second pH measuring device andallows determining, in case one or both of said pH measuring devices areoffline-devices, if one or both of the measured pH values may be flawedby an offset effect.

An early detection of calibration differences and/or offset effects mayallow recalibrating, repairing or exchanging the pH measuring devicebefore the medium is inoculated with the cell culture. The recalibratingmay include the option to calibrate the first pH measuring device suchthat it compensates for any offset effect. Thus, growing a cell culturein a bioreactor whose pH measuring device is calibrated differently thana pH measuring device of a reference bioreactor (or whose measured pHvalues are flawed by an offset effect) can be avoided and/or correctedfrom the beginning, thereby saving time and money that would be lost incase a calibration error of a pH measuring device results in a failureto reproduce the environmental conditions of the reference (“second”)bioreactor in the other (“first”) bioreactor.

In a further beneficial aspect, embodiments of the invention allowcomparing pH values of different bioreactors even in case the twocompared bioreactors and respective pH measuring devices are located farapart from each other, e.g. are located in different buildings ordifferent cities or even different countries. Instead of relying onstandardized, commercially available calibration solutions having adefined pH value, the CO2 value (which can easily and highly accuratelybe determined) is used as basis for comparing the measured pH values andfor identifying calibration differences and/or offset effects in a pHmeasurement. The measured first CO2 concentration and the first measuredpH value can be communicated to the comparison unit easily, e.g. via aninternet connection. It is not necessary that the first and second pHmeasuring devices are calibrated and compared at the same time. It isnot even necessary that the absolute pH value is correctly determined,Given a first pH value and a first CO2 value measured by a first pHmeasuring device in a first bioreactor at pH-CO2 equilibrium state asdescribed above, it is possible to determine, by measuring a second pHvalue and second CO2 value by a second pH measuring device in a secondbioreactor at pH-CO2 equilibrium state as described above, if the firstand second pH measuring devices are calibrated identically, even in casethe second pH value and second CO2 value were determined weeks or yearsbefore the first pH value and second CO2 values were observed.

In a further aspect, in case the first bioreactor shall be operatedbasically with the same environmental parameters like the secondbioreactor, determining the absence of calibration differences of thesecond and first pH measuring devices and determining the absence ofoffset effects in the pH measurements may be much more important than acorrect measurement of an absolute pH value. So even in case the secondbioreactor (whose operational parameters may provide a kind of“reference profile” for operating the first bioreactor) was operatedwith a wrongly calibrated pH measuring device and/or the second pH valueis influenced by an offset effect while the first pH measuring device(of the first bioreactor that shall be operated as specified in the“reference profile”) is calibrated correctly/has no offset effect,embodiments of the invention allow to identify a calibration differenceand/or the existence of an offset effect when measuring the second pHmeasuring device. The identification of the calibration differenceand/or offset effect when measuring the second or first pH value allowsan operator or an automated control system to take appropriate actionsto prevent growing of the cell culture in the first bioreactor underdifferent environmental parameters (in particular, under different pHvalues of the medium) than the cells of the second/reference bioreactor.

Thus, embodiments of the invention may enable accurate monitoring and/orcontrolling of the state of a bioreactor by identifying pH measuringdevice calibration differences and/or offset effects already at theinitialization phase of a bioreactor.

According to other embodiments, the first bioreactor comprises means formanually or automatically taking a sample of the medium in the firstbioreactor. During a time interval after filling the medium in the firstbioreactor and before adding the cell culture to the medium in the firstbioreactor, the method comprises keeping all openings of the samplingmeans closed.

This may be beneficial as a contamination of the medium of the firstbioreactor is avoided. Opening of the sampling means for determining thepH value in a reference solution of known pH value is not necessary anymore: calibration differences are identified via the CO2 values and thepH values are measured, according to embodiments of the invention, bythe pH measuring device in the bioreactor. This may be beneficial as therisk of infecting the bioreactor with unwanted microbes in the processof sampling is reduced and no offset effects are caused by the samplingprocess.

According to embodiments, the second and first CO2 concentrations of theoff gas of the respective bioreactors and the total gas influx rate areused for calculating a second CO2 off gas rate for the second bioreactorand for calculating a first CO2 off gas rate of the first bioreactor.Then, the second and first CO2 off gas rates are compared instead of thesecond and first CO2 concentrations. This approach may be applied incase the total off gas rates in the second and first bioreactor areidentical. Some CO2 off gas analyzers may measure a CO2 off gas rateinstead of a CO2 concentration and return a CO2 off gas rate to thecomparison unit. Provided that the total off gas rate of the twocompared bioreactors is identical, the CO2 off gas rate (“ACO”) of thetwo bioreactors may be compared instead of the two CO2 values and a pHmeter offset or calibration difference is detected if—given identicaltotal off gas rates and identical measured pH values the measured CO2off gas rates of the two bioreactors differ.

According to embodiments, the method is used for determining if thefirst pH measuring device is calibrated differently than the second pHmeasuring device. The first pH measuring device is at least partiallysurrounded by the medium within the first bioreactor. For example, thefirst pH measuring device may be a pH measuring device immersed in themedium of the first bioreactor. The first bioreactor lacks means formanually or automatically taking a sample of the medium in the firstbioreactor.

This may have the benefit of allowing using a bioreactor type thatprevents a contamination of the medium and/or the generation of offseteffects by taking samples for the purpose of measuring the pH value asan offline measurement.

According to other embodiments, the first bioreactor comprises means formanually or automatically taking a sample of the medium in the firstbioreactor. Said means may consist, for example, of an opening fortaking medium samples from the bioreactor manually, or may consist ofrobotic arms or drains for automatically or semi-automatically drawing asample. The method further comprises: during a time interval afterfilling the medium in the first bioreactor and before adding the cellculture to the medium in the first bioreactor, keeping all openings ofthe sampling means closed. This may prohibit contamination of the mediumin the bioreactor. As the method uses a first pH measuring deviceresiding inside the bioreactor in combination with a CO2 concentrationof the gas volume above the medium that can easily be measured withouttaking samples, embodiments of the invention allow for detectingcalibration differences without taking a sample of the bioreactor andthus without risking an infection.

According to embodiments, the method is used for determining if thefirst pH measuring device is calibrated differently than the second pHmeasuring device. The determination if the second and first pH measuringdevice are calibrated differently is performed while the first pHmeasuring device is at least partially surrounded with the medium in thefirst bioreactor (thus, by a pH measuring device completely or at leastpartially located inside the bioreactor) and without taking a sample ofthe medium of the first bioreactor for performing said determination.

According to embodiments, the determination is performed by using thesecond and first CO2 concentrations and the second and first pH valuesas the only data input for said determination. This may be advantageousas said parameter values can easily be obtained by performing online pHvalue and CO2 concentration measurements.

According to embodiments, the method further comprises performing anonline-measurement with the first pH measuring device for measuring thefirst pH-value, the first pH measuring device being at least partiallysurrounded with the medium in the first bioreactor.

In addition or alternatively, the method comprises performing anonline-measurement by a first CO2 sensor in the off gas of the firstbioreactor for providing the first CO2 concentration.

This method may in particular be used determining if the first pHmeasuring device is calibrated differently than the second pH measuringdevice (as the first pH measuring device is capable of performingonline-measurements, there is typically no offset effect and anydeviation from the result of the second pH measuring device are causedby calibration differences or, in case the second pH measuring device isan offline-measuring device, by offset effects of the second pHmeasuring device.

In many currently used bioreactor types, respective CO2 concentrationand pH measuring devices are already present and may easily be employednot only for bioreactor state monitoring and control purposes but alsofor the purpose of identifying calibration differences and offseteffects. It is neither necessary to draw medium samples for pHmeasurement nor to withdraw or reintroduce the pH measurement devicefrom or into a bioreactor. Thus, the risk of infecting the bioreactorwith undesired microbes is reduced. The method may allow identifyingcalibration differences of two pH-meters of two bioreactors withouttaking samples from the medium from any one of the two bioreactors.

Using a CO2 sensor that measures the CO2 concentration in the off gas ofa bioreactor may be advantageous, because CO2 off gas meters (“CO2 offgas analyzers”, “CO2 off gas sensors”) are non-invasive, do not need asampling, can be easily obtained in real time and deliver a value, theCO2 concentration in the off gas and/or the CO2 off gas rate. Thus, CO2off gas analyzers may give immediate response to intended or unintendedprocess changes in a bioreactor (in contrast to e.g. cell densities orcell counts). Further, off gas analyzers can be calibrated anytime anddo not have to be autoclaved.

According embodiments, the method comprises performing anonline-measurement with the second pH measuring device for measuring thesecond pH-value. The second pH measuring device is at least partiallysurrounded with the medium in the second bioreactor. In addition, oralternatively, the method comprises performing an online-measurement bya second CO2 sensor in the off gas of the second bioreactor forproviding the second CO2 concentration. This may have the advantage thatalso the pH and CO2 concentration values of the second (or “reference”)bioreactor may be gathered by means of online measurements, e.g. bymeans of an immersed, continuous pH-meter, thereby avoiding pH offseteffects and reducing the risk of infections caused by the samplingprocess.

According to embodiments, the method is used for determining if thefirst pH measuring device outputs a different pH value than the secondpH measuring device due to offset effects of a sampling processperformed for measuring the first pH value in a sample of the medium ofthe first bioreactor. The method further comprises performing anoffline-measurement with the first pH measuring device for measuring thefirst pH-value. The first pH measuring device is outside of the firstbioreactor and is at least partially surrounded with the medium in amedium sample of the first bioreactor.

Said features may be beneficial if a bioreactor type is used that istechnically equipped with offline measurement devices, in particularoffline pH measuring devices. In this context, any offset effectdetermined by a comparison of the second and first pH values and secondand first CO2 concentrations may be used for outputting a warning and/ormodifying the output of the first pH measuring device in a way that theoffset effect caused by the sampling process is compensated.

According to embodiments, the method comprises determining that thesecond and first pH measuring devices are calibrated differently ordetermining that the second and first pH measuring devices outputdifferent pH values due to offset effects of a sampling process in caseone of the following situations occurs:

-   -   the second and first CO2 concentrations are identical and the        second and first pH values differ from each other by more than a        threshold value; or    -   the second and first pH values are identical and the second and        first CO2 concentrations differ from each other by more than a        further threshold value; or    -   a second data value differs from a first data value by more than        a further threshold, the second data value being derived from        the second pH value and the second CO2 concentration, the first        data value being derived from the first pH value and the first        CO2 concentration. For example, in case the total off gas rates        are identical for two bioreactors whose pH measuring devices        shall be calibrated to allow a comparison of the pH value in the        media in both bioreactors, the CO2 off gas rates may be compared        instead of the CO2 concentrations.

According to embodiments, in case neither the pH value nor the CO2concentrations are identical, a control unit of a bioreactor monitoringand/or control system may modify the CO2 gas influx rate. Thereby, boththe CO2 concentration in the gas phase as the pH value in the medium maybe affected. As soon as either the first pH value is identical to thesecond pH value or the first CO2 concentration is identical to thesecond CO2 concentration, the above described determination isperformed.

According to embodiments, the method further comprises observing thatthe second and first CO2 concentration are identical and calibrating thefirst pH measuring device in a way that the first pH measuring deviceindicates the same pH value as the second pH measuring device.

For example, in case the second and first CO2 concentrations areidentical and the second and first pH value differ from each other by anamount “delta”, said “delta” pH offset value can be added to each pHvalue measured by the first pH measuring device in the future (i.e. at atime later than the first time). The resulting pH values output by thefirst pH-measuring device thus compensate for the pH offset caused by asampling process for measuring the second or first pH value and/orcompensate for any calibration differences between the second and firstpH measuring devices.

According to embodiments, a control unit of a system configured formonitoring and/or minimizing deviations of a state of the firstbioreactor from the state of the second bioreactor uses pH values of thefirst pH measuring device as an input. The minimization of the statedifferences and/or the comparison of the states of the two bioreactorsis performed by the comparison unit analyzing at least said inputparameters. The comparison unit may be a software, firmware and/orhardware-implemented piece of program logic, e.g. an application programrunning on an electronic data processing system. According toembodiments, the comparison unit is operatively coupled to the controlunit. For example, the comparison unit may be an integral part of thecontrol unit or may be an application program configured to interoperatewith the control unit. The control unit and the monitoring unit may behosted on the same or on different electronic data processing machines.

This may be advantageous as the use of a first pH measuring device thatis calibrated differently than the second pH measuring device and/orwhose pH values have an offset effect due to the sampling process can beavoided.

According to embodiments, the receiving of the second and first pHvalue, the receiving of the second and first CO2 concentration and thecomparison of said pH and CO2 concentration values is performed by thecomparison unit. In case of determining that the second and first pHmeasuring devices are calibrated differently, the comparison unit mayperform one or more of the following steps:

-   -   outputting a warning message;    -   automatically performing or triggering the performing of a        recalibration of the first pH measuring device;    -   automatically performing or triggering the performing of a        replacement of the first pH measuring device by a new first pH        measuring device.

Thus, an operator or an automated component of the first bioreactor isenabled to take appropriate actions, e.g. exchange or recalibrate thefirst pH measuring device and delay the inoculation of the firstbioreactor until the problem is fixed.

The comparison unit may be, for example, provided by or executed on anelectronic data processing apparatus or part thereof. The apparatuscomprises a processor, memory and electronic instructions storedthereon. Upon processing the instructions by the processor, the methodof embodiments of the invention is performed by the comparison unit. Insome embodiments, the comparison unit is operatively coupled to one ormore bioreactor monitoring and/or control application programs. Thecomparison unit may be an integral part of a system comprising the firstand optionally also the second bioreactor. The system, according to someembodiments, comprises further bioreactors whose state shall bemonitored and compared with the state of the second bioreactor.

According to embodiments, the comparison unit reads a medium-specificrelation from a data storage medium. The medium-specific relation isspecific for the medium in the second and in the first bioreactors andindicates a relation between the pH value of the medium and a respectivefraction of CO2 gas in a gas volume when said medium is in pH-CO2equilibrium state with said gas volume and lacks a cell culture. Then,the comparison unit uses the first CO2 concentration as input for themedium specific relation for calculating an absolute pH value expectedfor the medium in the first bioreactor in pH-CO2 equilibrium at thepredefined temperature and pressure and under the absence of a cellculture, Then, the comparison unit (or an operator using the calculationresult of the medium specific relation) configures the first pHmeasuring device such that the calculated absolute pH value is output bysaid first pH measuring device. For example, the first pH measuringdevice is calibrated such that in future pH measurements, the first pHmeasuring device outputs a sum of a measured first pH value and a deltapH, the delta pH being the difference between the measured first pHvalue and the expected pH value calculated by using the medium specificrelation.

The medium-specific relation can be, for example, an equationPPH_(M1)(CO2)=REL-M1(CO2) obtained by mathematically fitting multipleempirically determined pairs of a pH-value of the medium (M1) and arespectively measured fraction of CO2 gas in a gas volume above saidmedium. Thereby:

-   -   PPH_(M1)(CO2) is the predicted pH value in a medium (M1) when        said medium lacks a cell culture and is at pH-CO2 equilibrium        with a gas volume above said medium, said gas volume comprising        the CO2 concentration used as input parameter;    -   the CO2 is an input parameter value and represents the CO2        concentration in a gas volume above the medium (M1) in ph-CO2        equilibrium state under the absence of the cell culture;    -   REL-M1 is a set of one or more parameters connected by        operators.

The parameters are obtained e.g. by manually, automatically orsemi-automatically performing the following steps:

-   -   adjusting samples of the medium M1 lacking the cell culture to        multiple different pH values, thereby letting the samples reach        pH-CO2 equilibrium with the gas volume above the medium in the        respective sample,    -   determining the fraction of CO2 gas in a in respective gas        volume being in ph-CO2 equilibrium with the medium in the        samples,    -   plotting the determined CO2 gas fractions against the respective        equilibrium pH values of the samples,    -   fitting a curve in the plotted values and deriving the        parameters of the medium-specific relation from the fitted        curve.

Thus, the medium-specific relation may be identified empirically, e.g.before the second or first bioreactor is inoculated with the referencecell culture.

According to some embodiments, the medium specific relation is obtainedby filling a bioreactor, e.g. the second bioreactor, with the medium,whereby the medium does not comprise the cell culture cells, and settingthe temperature and pressure of the second bioreactor to predefinedvalues, e.g. 20° C. and standard atmospheric pressure. The medium in thebioreactor used for empirically determining the medium-specific relationis referred herein also as one of the samples whose pH value is to beset.

Then, the medium may be set to different pH values by increasing ordecreasing the CO2 concentration in the gas volume above the medium viaa modified CO2 gas influx rate, and after some time (typically minutesor hours) when the medium has equilibrated (reached pH-CO2 equilibriumstate at the given pH and the predefined temperature and pressure), theCO2 concentration in the gas volume above said medium (which correlateswith the CO2 partial pressure at said equilibrium state) is measured.Said measuring is performed e.g. by analyzing the CO2 concentration inthe gas volume above the medium or via the CO2 volume fraction in theoff gas. The acquired pairs of equilibrium pH-values and CO2concentrations (or CO2 off gas values) measured in the sample(bioreactor or aliquot) are plotted, i.e., represented in a coordinatesystem. The plotting may be performed automatically by an electronicdata processing system which may in addition output the plot in the formof a paper-based printout and/or a plot displayed on a display screen.The plotting may also be performed manually. A curve is fittedautomatically or manually to said plot and parameters being descriptiveof said fitted curve are computed. The parameters define themedium-specific relation of pH-value and CO2 concentration of a gasvolume above said medium when said medium is in pH-CO2 equilibrium at aparticular pH value. The pH value is preferably set by adjusting the CO2influx rate, not by adding basic or acid substances in order to avoid amodification of the composition of the medium.

After having computed the parameters, the medium specific relation maybe transferred to the comparison unit, e.g. via a graphical userinterface allowing a user to enter the relation manually, via a portablestorage medium or via a network connection.

According to other embodiments, the medium-specific relation is obtainedby creating multiple samples in the form of aliquots of said medium,each sample having a different pH value. The samples are left at apredefined temperature and pressure for some time to allow pH-CO2equilibration between the gas and the liquid medium in each sample.

The samples can be obtained sequentially, e.g. by changing the pH valueof a single sample and performing sequential measurements, or can beobtained by creating multiple samples of said medium in parallel, eachaliquot being set to a different pH value by modifying the CO2concentration of the gas volume above the medium. The sample can befilled into any container allowing the setting and measuring of acurrent pH value and allowing the modification of the CO2 concentrationand the measuring of a CO2 concentration or CO2 off gas rate atequilibrium state. Preferentially, the pH value in the respectivesamples is set by adapting the CO2 influx rate and thus the CO2concentration in the gas phase of a bioreactor in a way that the pHvalue adapts accordingly. This may be advantageous as a modification ofthe composition of the medium (except of the dissociation product ofsolved CO2) does not change when using CO2 rather than acids or basesfor adapting the pH.

According to some embodiments, the equation PPH_(M1)(CO2)=REL-M1(CO2) isa linear equation according to PPH_(M1)(CO2)=a1×pH+a2. In this case, theparameters a1 and a2 are the parameters derived from the fitted curve.The PPH_(M1)(CO2) indicates the predicted pH value in a medium inequilibrium state with a gas volume having the particular CO2concentration used as input to said equation.

According to other embodiments, the equation PPH_(M1)(CO2)=REL-M1(CO2)is a polynomial equation according to PPH_(M1)(CO2)=b1×pH²+b2×pH+b3. Inthis case, the parameters b1, b2 and b3 are the parameters derived fromthe fitted curve.

Empirically determining the medium-specific relation and thecorresponding medium-specific parameters may have the beneficial effectthat even in case the exact composition of the medium is not known(which is commonly the case for many media in the market), the impact ofa particular pH value on the equilibrium CO2 concentration in an airvolume in pH-CO2 equilibrium with said medium can be determinedexperimentally. Thus, an absolute pH value can be calculated and can beused for calibrating a pH measuring device also when using media typeswhose composition is not known.

According to embodiments, the cells of the cell cultures are prokaryoticor eukaryotic cells, in particular mammalian cell culture cells.

According to embodiments, the second bioreactor differs from the firstbioreactor in respect to one or more of the following features:

-   -   a) the gas volume in the bioreactor,    -   b) the medium volume in the bioreactor,    -   c) the Reynolds number of the bioreactor,    -   d) the Newton number of the bioreactor,    -   e) the dimensions of the bioreactor,    -   f) geometrical features of the bioreactor and/or bioreactor        baffles,    -   g) the stirrer configuration,    -   h) the stirring rate,    -   i) the volumetric mass transfer coefficient for oxygen (kLa) of        the bioreactor,    -   j) total gas influx rate and/or O2 influx rate and/or N2 influx        rate and/or CO2 influx rate,    -   k) power input,    -   l) pressure in the bioreactor,    -   m) gas bubble hold time in the medium,    -   n) gas bubble size and distribution in the medium,    -   o) surface speed,    -   p) a parameter calculated as a derivative from one or more of        the parameters a)-o);    -   q) the geographic location of the two bioreactors (e.g.        different countries, cities, buildings)

The “power input” parameter as used herein specifies the amount of powerinput of a stirrer of a bioreactor. Different stirrer configurations canhave different power inputs at identical agitation or identical tipspeeds. Power input at identical stirrer speeds may depend on theviscosity of the medium.

By comparing CO2 off gas concentrations, the calibration state of pHmeasuring devices of two bioreactors can be easily compared: identicalCO2 concentrations in the off gas indicate identical calibration of pHmeasuring devices of different bioreactors even in case said bioreactorshave different Reynolds and/or Newton numbers, have a different speed orconfiguration of the stirrer or the like.

According to embodiments, the comparison unit receives a third CO2concentration and a third pH value. The third CO2 concentration is a CO2concentration of a third gas volume above the medium in the secondbioreactor. The third CO2 concentration and the third pH value aremeasured at a third time. The third time is a time when the medium inthe second bioreactor is in pH-CO2 equilibrium state at a predefinedtemperature and pressure with the third gas volume and after saidequilibrium state is modified by the metabolism of the cell culture inthe second bioreactor. The third pH value is a measured value providedby the second pH measuring device.

Then, the comparison unit receives a fourth CO2 concentration and afourth pH value. The fourth CO2 concentration is a CO2 concentration ofa fourth gas volume above the medium in the first bioreactor. The fourthCO2 concentration and the fourth pH value are measured at a fourth time.The fourth time is a time when the medium in the first bioreactor is inpH-CO2 equilibrium state at the predefined temperature and pressure withthe first gas volume and after said equilibrium state is modified by themetabolism of the cell culture in the first bioreactor. For example,after some hours or even days, some cells start excreting substances,e.g. lactate, into the medium which change the pH and/or composition ofthe medium. The fourth pH value is a measured value provided by thefirst pH measuring device. The lapsed time between the third time andthe inoculation of the second bioreactor is identical to the lapsed timebetween the fourth time and the inoculation of the first bioreactor.

In addition, the comparison unit receives a measured second oxygenuptake rate of the cell culture in the second bioreactor at the thirdtime and receives a measured first oxygen uptake rate of the cellculture in the first bioreactor at the fourth time.

In case the second and first oxygen uptake rates are identical, thecomparison unit compares the third and fourth pH values and compares thethird and fourth CO2 concentrations for determining if the second andfirst pH measuring devices are calibrated differently or for determiningif the second and first pH measuring devices output different pH valuesdue to offset effects of a sampling process performed for measuring thethird or fourth pH value in a sample of the medium of a respective oneof the second and first bioreactors.

The second and first pH measuring devices are determined to becalibrated differently or are determined to output different pH valuesdue to offset effects of a sampling process in case one of the followingsituations occurs:

-   -   the third and fourth CO2 concentrations are identical and the        third and fourth pH values differ from each other by more than a        threshold value; or    -   the third and fourth pH values are identical and the third and        fourth CO2 concentrations differ from each other by more than a        further threshold value

Embodiments of the invention assume that in case the temperature andpressure is identical in the second and first bioreactor and in case inaddition the oxygen uptake rate is identical, then differences in themeasured pH values result from calibration errors or offset effects of asampling process for measuring a pH value.

Said features may be particularly advantageous as they allow todetermine if two pH measuring devices are calibrated differently or showsampling offset effects even in case the metabolism of the cells hasstarted modifying the pH-CO2 equilibrium state of a bioreactor, e.g. byexcreting lactate (and without taking samples for offline pHmeasurements). It was observed that often the oxygen uptake rate ofcells correlates with the state of a particular cell culture, so in casethe OUR of the cell cultures in two bioreactors is identical and alsothe CO2 concentrations in the off gas, temperature and pressure areidentical, any observed pH differences are caused by calibration errorsor sampling-based offset effects.

In a further aspect, the invention relates to a comparison unitconfigured for:

-   -   receiving a second CO2 concentration and a second pH value, the        second CO2 concentration being a CO2 concentration of a second        gas volume above a medium in the second bioreactor, the second        CO2 concentration and the second pH value being measured at a        second time, the second time being a time when the medium in the        second bioreactor is in pH-CO2 equilibrium state at a predefined        temperature and pressure with the second gas volume and before        said equilibrium state is modified by the metabolism of a cell        culture in the second bioreactor, the second pH value being a        measured value provided by a second pH measuring device        operatively coupled to a second bioreactor;    -   receiving a first CO2 concentration and a first pH value, the        first CO2 concentration being a CO2 concentration of a first gas        volume above a medium in the first bioreactor, the first CO2        concentration and the first pH value being measured at a first        time, the first time being a time when the medium in the first        bioreactor is in pH-CO2 equilibrium state at the predefined        temperature and pressure with the first gas volume and before        said equilibrium state is modified by the metabolism of a cell        culture in the first bioreactor, the first pH value being a        measured value provided by a first pH measuring device        operatively coupled to a first bioreactor;    -   comparing the second and first pH values and CO2 concentrations        for determining if the second and first pH measuring devices are        calibrated differently or for determining if the second and        first pH measuring devices output different pH values due to        offset effects of a sampling process performed for measuring the        second or first pH value in a sample of the medium of a        respective one of the second and first bioreactors.

In a further aspect the invention relates to a system configured formonitoring and/or minimizing deviations of a state of the firstbioreactor from the state of the second bioreactor. The system comprisesa control unit for monitoring and/or controlling at least the first andoptionally also the second and one or more further bioreactors andcomprises a comparison unit according to any one of the embodimentsdescribed herein. The system further comprises at least the firstbioreactor and the first pH measuring device. The control unit isconfigured to monitor and/or control a state of a cell culture at leastin the first bioreactor, thereby using pH values repeatedly measured bythe first pH measuring device.

According to embodiments, the system further comprises the secondbioreactor, whereby second and first bioreactors are located indifferent geographic regions and optionally coupled to the comparisonunit via a network, e.g. the internet, for communicating the CO2concentrations and pH values.

According to embodiments, the second time is a time before the secondbioreactor is inoculated with a cell culture. The second time may alsobe a time at or after inoculation of the second bioreactor with the cellculture and before the metabolism of said cell culture modifies the pHvalue of the medium in the second bioreactor.

The first time is a time before the first bioreactor is inoculated witha cell culture. Alternatively, the first time is a time at or afterinoculation of the first bioreactor with the cell culture and before themetabolism of said cell culture modifies the pH value of the medium inthe first bioreactor.

A “profile” as used herein is a representation of the variation in aparameter value versus time.

A “tank” as used herein is a container for holding, transporting, orstoring liquids. A tank can be, for example, a bioreactor or a harvestor transport tank comprising the medium, cell culture and reactionproducts of a bioreactor. A tank may also be a calibration box that isfilled with cell-free medium and is used for calibrating a pH measuringdevice.

A “calibration box” as used herein is a tank with a known medium,whereby the tank is operatively coupled to a CO2 sensor and isconfigured for temporarily housing one or more pH measuring devices forcalibrating the pH measuring devices using the CO2 offgas concentrationmeasured by the sensor. For example, the calibration box can be abioreactor that is currently not used for growing a cell culture but isused solely or predominantly for calibrating pH measuring devices.Alternatively, the calibration box may be a special purpose container,in particular a portable container that can be carried by a person todifferent places for calibrating the pH meters in differentlaboratories. The calibration box comprises a medium with knownproperties (current temperature, pressure, known medium specificrelation or known composition in the case of purely bicarbonate-bufferedmedia) and comprises a CO2 sensor in the gas volume above the medium orin the offgas of the calibration box. It further comprises an openingfor easily inserting and removing a pH measuring device and may compriseone or more fixing devices for temporarily fixing the one or more pHmeasuring devices in the calibration box such that they are at leastpartially surrounded by the medium in the calibration box.

A “predefined” temperature and pressure as used herein specifies to atemperature and pressure that is controlled or at least known by anoperator of the first and/or second tank or that is controlled by or atleast “known” by a program logic configured to operate the first and/orsecond tank or the pH measuring devices contained therein. Thus, the“predefined” temperature and pressure may also be referred to as “given”temperature and pressure. It may be necessary to ensure that the firstand second pH values and first and second CO2 values are measured at thesame, given temperature and pressure.

A “comparison unit” as used herein is a piece of program logic, e.g. anapplication program or module, a computer chip or another piece ofhardware or firmware that is configured for receiving and processing oneor more measured pH values and one or more measured CO2 concentrationsin the off gas of a tank for determining if a pH measuring erroroccurred. For example, the comparison unit may be a program module thatis part of or interoperates with a calibration software or bioreactormonitoring or control software.

A pH measuring device being “operatively coupled” to a tank can be, forexample, a pH measuring device that is permanently or temporarilylocated within the tank and is configured to perform on-line pHmeasurements.

A “bioreactor” as used herein is a vessel in which a chemical process iscarried out which involves organisms or biochemically active substancesderived from such organisms. This process can be, for example, aerobicor anaerobic. A plurality of different bioreactor types exist which varyin shape (e.g. cylindrical or other), size (e.g., milliliters, liters tocubic meters) and material (stainless steel, glass, plastic, etc.).According to embodiments, the bioreactor is adapted for growing cells ortissue in cell cultures. Depending on the embodiment and/or on the modeof operation, a bioreactor may be a batch bioreactor, fed batchbioreactor or continuous bioreactor (e.g. a continuous stirred-tankreactor model). An example of a continuous bioreactor is the chemostat.

An “Online-measurement” as used herein is a process of obtaining ameasurement value being descriptive of state features of a bioreactor orof a cell culture contained therein, whereby the duration required forperforming the measurement is shorter than the time during which saidfeatures significantly change. A significant change can be a change bymore than a predefined threshold value. For example, a change by morethan 5% may be considered as a significant change. The threshold mayvary for different features. Online-measurements may allow controlling abioreactor in real time.

An “Offline-measurement” is a process of obtaining a measurement valuebeing descriptive of state features of a bioreactor or of a cell culturecontained therein, whereby the duration required for performing themeasurement is longer than the time during which said features cansignificantly change. A significant change can be a change by more thana predefined threshold value. A typical example for anoffline-measurement is the automated, semi-automated or manual samplingof a the medium e.g. for measuring a current pH value. Offlinemeasurements are based on a discontinuous sampling process. As thebioreactor features may meanwhile have changed since the sample wastaken, controlling the bioreactor based on offline-measurement datatends to be of low quality due to significant latency times between themoment of measurement and the moment of performing a respective controloperation.

A significant change can be a change by more than a predefined thresholdvalue, for example 2% or any other percentage value, depending on therespective state feature. A typical example for an offline-measurementis the automated, semi-automated or manual sampling of a probe of themedium e.g. for measuring a current pH value for calibrating a pHmeasuring device when initiating a bioreactor.

A discontinuous sampling process for obtaining the measurement valuefrom a sample may have the disadvantage that the bioreactor features maymeanwhile have changed. Thus, controlling the bioreactor based onoffline-measurement data tends to be of low quality due to significantlatency times between the moment of measurement and the moment ofperforming a respective control operation.

A “pH measuring device” or “pH meter” as used herein is a device and/orsubstance used for measuring a current pH value in a medium. A pH metercan be, for example, a pH indicator (like phenolphthalein)—in form of asolution or pH strips—or a potentiometric apparatus. According topreferred embodiments, the pH meter is a continuous pH meter, i.e., a pHmeter capable of continuously and repeatedly measuring the pH of themedium of a bioreactor without having to draw samples and without havingto insert said pH meter in the medium for each individual measurement.For example, a pH meter can be a precise voltmeter, connected to themedium and to a reference electrode, and scaled in such a way that itdisplays not the measured potential, but ready pH value. Preferentially,the pH meter is immersed in the medium and is used for repeatedlymeasuring the current pH value in the medium during the whole time whilecultivating cells in the bioreactor. For example, the pH meter maymeasure a current pH value every minute, or every 30 minutes, or everyhour. In typical today's pH meter reference electrode is built into thepH electrode, which makes the device compact.

A “CO2 measuring device”, “CO2 sensor”, “CO2 meter” or “CO2 analyzer” asused herein is a device used for measuring a current CO2 concentrationin a gas volume, e.g. the gas volume above the medium of a bioreactor orthe off gas of a bioreactor.

According to embodiments, the current CO2 concentration of the secondand/or first bioreactor is measured by a continuous CO2 off gas meter,i.e., a device capable of measuring the current CO2 concentration in theoff gas of a bioreactor repeatedly without having to insert or replace ahardware module into the bioreactor or its connected off gas pipe orpipes for each CO2 concentration measurement. Using continuous pHmeasuring devices and/or continuous CO2 off gas meters may beadvantageous as the respective measurements can be performed easily andrepeatedly without causing offset effects and/or without the need totake a sample of the medium. Many existing bioreactors already compriseone or more immersed pH-meters and/or comprise or are coupled withmeasurement devices capable of measuring the CO2 concentration and/orthe CO2 off gas rate.

Depending on the embodiment, the bioreactor (or reference bioreactor)comprises a single gas inflow line or pipe or multiple gas inflow linesor pipes. For example, a single gas inflow line or pipe may be used fordelivering environmental air or (already expanded) compressed air fromspecial suppliers into the bioreactor (reference bioreactor). Saidenvironmental air or compressed air may consist of a mixture of gasses,in particular N2, O2 and CO2 that is typical for the earth's atmosphereor has a different composition. In addition or alternatively, the singlegas inflow line or pipe or any of the other gas inflow lines or pipesmay be used for delivering individual gases such as N2, O2 and CO2 tothe bioreactor, e.g. to control the cell growth.

According to embodiments, one or each of the two bioreactorsrespectively comprises a microsparger for generating very finelydispersed gas bubbles from the inflowing gas for accelerating theestablishment of a pH-CO2 equilibrium between the medium and the gasvolume in the bioreactor. For example, a microsparger may be used for aninflux gas mix or for each individual influx gas component separately.In addition, or alternatively, one or each of the two bioreactors isconfigured and operated such that the carbon dioxide and one or moreother gases (e.g. nitrogen, oxygen and/or air) are added togethersimultaneously to the bioreactor as a gas mix. For example, all influxgases may be input to the bioreactor as gas mix, e.g. via a submersedpipe opening or a microsparger.

Preferentially, all process gases are input to the bioreactor via amicrosparger and/or in the form of a gas mix in case the volume of thebioreactor is below a threshold volume of e.g. 400 liter or e.g. 200liter.

According to embodiments, the aeration rate and bubble size of theinflux gases in the medium of the bioreactor is chosen such that all gasbubbles reach pH-CO2 equilibrium with the medium before leaving thebioreactor or are dissolved completely in the medium.

Said features may be advantageous as they ensure that the gas bubblesreach equilibrium state before their gas content leaves the bioreactor:a microsparger generates very finely dispersed gas bubbles of theinflowing gas, thereby accelerating the establishment of a pH-CO2equilibrium between the medium and the gas volume in the bioreactor.Inputting the CO2 gas as a gas mix avoids the situation that the CO2transition rate from a pure CO2 gas bubble into the medium is largerthan the CO2 transition rate from the medium to e.g. air or N2 bubbles(the transition rate may depend on the amount of CO2 concentrationdifference between medium and different types of bubbles). Thus, saidmeasures ensure the comparability of the state of bioreactors over awide range of bioreactor volumes, including volumes below e.g. 400liter.

A “profile” as used herein is a representation of the variation in aparameter value versus time.

The “pH-CO2 equilibrium” indicates a state of a system comprising anaqueous solution (e.g. a cell culture medium) and an air volume abovesaid solution (e.g. the gas volume in a bioreactor) whose pH value andCO2 partial pressure are in chemical equilibrium according to theHenderson-Hasselbalch equation. The CO2 partial pressure corresponds tothe fraction of CO2 gas in the total gas volume above the medium. TheHenderson-Hasselbalch equation describes the relationship of pH as ameasure of acidity with the acid dissociation constant (pKa), inbiological and chemical systems. If a gas comprising CO₂ is in contactwith an aqueous liquid, e.g. a culture medium, at least a small fractionof the CO2 dissolves in said liquid. At room temperature, for example,the solubility of carbon dioxide is about 90 cm³ of CO₂ per 100 ml water(c_(l)/c_(g)=0.8). Any water-soluble gas becomes more soluble as thetemperature decreases. A small fraction (ca. 0.2-1%) of the dissolvedCO₂ is converted to H₂CO₃. Most of the CO₂ remains as solvated molecularCO₂. This process can be described by the following formulas:

Carbonic acid (H2CO3) equilibrium:[CO2]×[H2O]←→[H2CO3]←→[H+]×[HCO3−][H+]×[HCO3−]=K×[CO2]×[H2O],wherein K=equilibrium constantpH=pK+log([HCO3−]/[CO2])

A “CO2 volume fraction” as used herein is the fraction of CO2 gas in atotal gas volume. The unit may be, for example, Vol. %. It is alsoreferred to as “CO2 concentration” of a gas volume, the concentrationbeing specified in Vol. %.

A “medium” or “cell culture medium” is a liquid or gel designed tosupport the cultivation and typically the growth of microorganisms orcells, or small plants like the moss Physcomitrella. There are differentmedia for growing different types of cells. Typically, a medium is awater-based solution comprising mixture of one or more substances suchas salt(s), carbohydrates, trace elements, peptides and/or proteins.There exist a plurality of different media on the market, e.g. for cellculture of specific cell types derived from plants or animals, andmicrobiological culture for growing microorganisms, such as bacteria oryeast. A medium may be, for example, a nutrient medium, e.g. an LBmedium (Lysogeny Broth), a minimal medium, a selective medium, adifferential medium, or an enriched medium. Some media may require a CO2environment of e.g. 5-10% CO2 to maintain physiological pH.

According to some embodiments, the expression “two media being the same”implies that the two media (e.g. the medium in the reference bioreactoron the one hand and the medium in the monitored and/or controlledbioreactor on the other hand) comprise—given a particular pressure,temperature and CO2 concentration in the gas volume above saidmedium—the same composition and concentration of organic and inorganiccompounds and solvents and/or have been manufactured using the samemanufacturing protocols and conditions within the context of measuringaccuracy.

According to some embodiments, said expression implies that the twomedia can differ in respect to any of said criteria (composition,concentration, manufacturing protocol) only in so far as said difference(at a given temperature, pressure and CO2 concentration in the gasvolume above said medium) has no or approximately no impact on thepH-CO2 equilibrium of said medium at a plurality of different pH valuesand in so far as the medium-specific relations derived empirically fromsaid two media respectively are identical.

To “cultivate a cell culture” as used herein typically means that thecells culture is grown, i.e., the number of the cells of the cellculture increases. In some occasions, however, the number of cells mayalso stagnate or even decline.

In the following embodiments of the invention are explained in greaterdetail, by way of example only, making reference to the drawings inwhich:

FIG. 1 shows a block diagram of a system for monitoring and/orcontrolling one or more bioreactors configured to detect a pH measuringdevice calibration deviation or a pH measurement offset effect;

FIG. 2 shows a flowchart of methods for detecting pH measuring devicecalibration deviations or pH measurement offset effects;

FIG. 3 shows components of a bioreactor;

FIG. 4 shows diagrams illustrating the dependence of the CO2concentration in the off gas of a bioreactor from the pH value;

FIG. 5 shows a plot used for obtaining a medium-specific pH-CO2concentration relation;

FIG. 6 shows pH values of four different bioreactors measured byrespective pH meters while growing a cell culture in the respectivebioreactors;

FIG. 7 shows the CO2 fraction measured in the off gas of each of thefour bioreactors;

FIG. 8 a is a diagram showing CO2-off gas-derived state profiles of twobioreactors and of a reference bioreactor;

FIG. 8 b is a diagram showing the state profile differences of the twobioreactors of FIG. 8 a to said reference bioreactor.

FIG. 9 is a plot that illustrates that the sampling process results in adeviation of the pH value measured in the sample from the pH value inthe tank and that the way the pH measuring devices are calibrated alsohas an impact on the measured pH value.

DETAILED DESCRIPTION

FIG. 1 shows a block diagram of a system 100 comprising a control unit132 for monitoring and/or controlling one or more bioreactors. Thesystem 100 comprises a comparison unit 130 for comparing measured pHvalues and CO2 concentrations received via an interface 128 from two ormore bioreactors. In the following, embodiments of the invention will bedescribed by making reference to a corresponding method for identifyingcalibration differences and pH measurement sampling offset effects asindicated in the flow chart of FIG. 2

FIG. 1 shows a system 100 allowing for real-time and accurate comparisonof pH values measured by pH measuring devices of two or more bioreactorsand of CO2 concentrations measured by off gas analyzers of respectivebioreactors for identifying calibration differences and offset effectsin two compared bioreactors immediately, without having to take mediumsamples of one of the bioreactors and without the need to use a“standard” solution of a known pH value for determining calibrationerrors or offset effects.

The system 100 comprises a processor 110, a main memory 112 and anon-transitory storage medium 114. The storage medium comprises computerreadable instructions which, when executed by the processor 110 causethe processor to perform a method for automatically monitoring and/orcontrolling one or more bioreactors 102, 104, 106 as described forembodiments of the invention.

The storage medium 114 comprises at least one data structure 136, e.g. afile or a database record, being indicative of a pH-CO2-concentrationrelation that is particular for the medium M1 contained in any of thebioreactors 102, 104, 106.

In addition, the storage medium may comprise medium-specific relations138 of other cell culture media M2. The medium-specific relations 136,138 may be received via a data communication interface 120, e.g. anetwork interface, an USB-port, a CDROM drive or the like.

The system 100 may further comprise an interface 126 for dynamicallyreceiving current measurement values from one or more monitored and/orcontrolled bioreactors 102, 104, 106. The interface 126 may also be anetwork interface, e.g. the Internet, or an Intranet. The measurementvalues are in particular a current pH value and a current CO2concentration measured in the off gas of the respective bioreactor. Acomparison unit 130 uses the received measurement values received fromthe monitored and/or controlled bioreactors 102, 104, 106 fordetermining if the respective pH measuring devices are calibrated in thesame way and are free of offset effects which prohibit a correctcomparison of measured pH values received from different bioreactors.Optionally, the comparison unit 130 also uses the medium-specificrelation 136 of the medium M1 as input in order to determine if a pHmeasuring device outputs a correct absolute pH value.

The first bioreactor 104 is initialized by filling the first bioreactorwith the cell-free medium M1 and by starting continuously adding gas,e.g. by transporting environmental air and/or its individual components(N2, O2 and/or CO2) to the bioreactor and optionally also by startingcontinuously adding liquids (the cell-free medium, optionally additionalliquids such as feed, etc.). In addition, the stirrers may be started.The first bioreactor thereby is operated at a temperature and pressurethat is identical to the temperature and pressure used for initiatingthe second bioreactor.

After some time (typically minutes or hours), the medium in the firstbioreactor and the air volume in the first bioreactor above the mediumwill have reached pH-CO2 equilibrium state and the first pH and CO2concentration values are measured in the medium and off gas of the firstbioreactor. In order to set the medium in the first bioreactor to aparticular pH value, the CO2 influx rate to the first bioreactor may bemodified accordingly, because the CO2 concentration in the gas volumehas an impact on the pH value of the medium.

In a second step 202, the comparison unit 130 receives a second CO2concentration CO2-R-M-ti and a second pH value pH_(R-M-ti). The secondCO2 concentration is a CO2 concentration of a second gas volume above amedium in a second bioreactor 102. The second CO2 concentration and thesecond pH value being measured at a second time ti. The second time is atime when the medium in the second bioreactor is in pH-CO2 equilibriumstate at a predefined temperature and pressure (e.g. 20° C. and normalatmospheric pressure) with the second gas volume and before saidequilibrium state is modified by the metabolism of a cell culture in thesecond bioreactor. For example, the second time ti is a time before thebioreactor 102 is inoculated with the cell culture or a time shortlyafter the inoculation so the metabolism of the cells does not have animpact on the pH-CO2 equilibrium in the bioreactor 102 yet. The secondpH value is a measured value provided by a second offline or online pHmeasuring device 142 operatively coupled to the second bioreactor 102.In the depicted example, the second pH measuring device is an online pHmeter immersed in the medium M1 of the second bioreactor 102.

In a next step 204, the comparison unit receives a first CO2concentration CO2_(B1-M-ti) and a first pH value pH_(B1-M-ti). The firstCO2 concentration is a CO2 concentration of a first gas volume above amedium in a first bioreactor which may be measured, for example, in theoff gas of the first bioreactor 104. The first CO2 concentration and thefirst pH value are measured at a first time. The first time is a timewhen the medium in the first bioreactor 104 is in pH-CO2 equilibriumstate at the predefined temperature and pressure with the first gasvolume and before said equilibrium state is modified by the metabolismof a cell culture in the first bioreactor.

For example, a CO2 analyzer device 122, also referred to as “carbondioxide sensor” may be used for repeatedly measuring the concentrationof CO2 in the off gas. Common examples for CO2 sensors are infrared gassensors (NDIR) and chemical gas sensors. NDIR sensors are spectroscopicsensors to detect CO2 in a gaseous environment by its characteristicabsorption. Alternatively, the CO2 sensor may be amicroelectromechanical sensor.

The first pH value is a measured value provided by a first pH measuringdevice 108 operatively coupled to the first bioreactor 104. The mediumin the second and in the first bioreactor are the same.

In some embodiments, the second bioreactor 102, also referred to as“reference bioreactor”, is used for growing a cell culture days, weeksor even years before the first bioreactor 104 is inoculated in order togrow a cell culture under basically the same conditions as in thereference bioreactor before. In this case, the second and first time maylie years apart, but respectively represent a time at which therespective bioreactor is initialized and does not (yet) comprise a cellculture having an impact on the pH-CO2 equilibrium. In this case, thesecond pH value and the second CO2 concentration are measured before thefirst pH value and first CO2 concentration is measured. In otherembodiments, the second and the first bioreactors are operated inparallel and the second and first pH and CO2 values may be measured andreceived by the comparison unit approximately at the same time.

In step 206, the comparison unit compares the second and first pH valuesand CO2 concentrations for determining if the second and first pHmeasuring devices are calibrated differently.

The comparison unit will determine that the second and first pHmeasuring devices are calibrated differently or that at least one ofsaid devices is affected by an offset effect (caused by the samplingprocedure) in case:

-   -   the second and first CO2 concentrations are identical and the        second and first pH values differ from each other by more than a        threshold value; or    -   the second and first pH values are identical and the second and        first CO2 concentrations differ from each other by more than a        further threshold value

In case the second and the first pH measurement devices are both onlinemeasuring devices, there do not exist any offset effects caused by asampling process. In this case, the comparison unit determines thatthere is a calibration difference between the second and the first pHmeasuring device and may output a warning message and/or a delta of thesecond and first pH values on the display 134. The display device may bee.g. a computer monitor or a monitor of a smartphone. Thus, an operatormay prohibit inoculation of the first bioreactor and perform arecalibration or an exchange of the first pH measuring device. It isalso possible that the moderator or the comparison unit reconfigures thefirst pH measuring device in a way that it outputs a value beingidentical to the second pH value for the medium in the first bioreactorwhose equilibrium CO2 concentration in the (off)gas phase was determinedto be identical to the equilibrium CO2 concentration in the (off)gasphase of the second bioreactor. In some embodiments, the control unit132 controls one or more parameters of one or more of the bioreactors102, 104, 106 such that the difference of environmental conditions forthe cells in the first bioreactor to the environmental conditions forthe cells in the second (reference) bioreactor is minimized. The controlunit can be, for example, a software and/or hardware module beingoperatively coupled to the comparison unit 130 for receiving the resultsof the comparison. The control unit is capable of controlling theconfiguration and operation of one or more engineering processes andparameters. For example, the control unit 132 may be operable toincrease or decrease the influx of liquids having an impact on the pHvalue, e.g. may increase or decrease the influx of a citric acid or of a1 M NaOH solution and/or may increase or decrease CO2 gas influx formodifying the pH value in the medium of a bioreactor.

The medium M1 can be, for example, Kaighn's Modification of Ham's F-12Medium comprising, for example, putrescine, thymidine, hypoxanthine,zinc, and higher levels of all amino acids and sodium pyruvate. Theseadditions allow the medium to be supplemented with very low levels ofserum or defined components, for some cell types. Ham's F-12K (Kaighn's)Medium contains no proteins or growth factors, and is therefore oftensupplemented with growth factors and Fetal Bovine Serum (FBS) that maybe optimized for a particular cell line. Ham's F-12K (Kaighn's) Mediumuses a sodium bicarbonate buffer system (2.5 g/L). The medium M2 may bean LB medium, and there may exist reference profiles for a plurality ofother media M3, M4, e.g. for cultivating bacteria or plants for avariety of purposes and corresponding “projects”.

The system 100 comprises the comparison unit and one or more bioreactors104 106 which are to be monitored and/or controlled by a control unit132 operatively coupled to the one or more bioreactors. As can beinferred from FIG. 1 , the dimensions and engineering parameters(stirring rate and configuration, bubble size, dimension, medium volumeetc.) of the monitored or controlled bioreactors may differ from eachother and/or may differ from the respective parameters of the referencebioreactor. The bioreactors 102, 104, 106 may be located at differentgeographic regions. One or more of the bioreactors send monitoring data(current pH and CO2 concentration in the off gas and/or CO2 off gasrates) to the comparison unit 130 and/or the control unit 132 andoptionally also receive control data from the control unit 132 orreceive control data for reconfiguring, recalibrating or exchanging a pHmeasuring device from the comparison unit. The reference bioreactor maybut does not have to be coupled to the system 100. It is sufficient thatthe second pH and CO2 concentration values gathered from the referencebioreactor are accessible by the comparison unit 130 when initiating thefirst bioreactor (or any further bioreactor 106 whose pH measuringdevice shall be calibrated in the same way as the second pH measuringdevice and should be free of any offset effects in respect to the pHvalues measured by the second pH measuring device 142). The secondbioreactor and each of the first bioreactors 104, 106 comprise the samemedium M1 and are inoculated with the same type of cell culture.

Preferentially, the monitored and/or controlled bioreactor 104, 106 atleast at the time point of initialization is operated under the sametemperature and pressure as the reference bioreactor. However, it ispossible that while operating the bioreactor 104, 106, the temperatureand/or pressure is modified in order to minimize state differences inrespect to the cell culture state in the reference bioreactor.

FIG. 3 shows an embodiment of a bioreactor 102, 104, 106. The bioreactoris coupled to a second pipe or hose for transferring fresh medium andoptionally one or more further liquids into the bioreactor. Thebioreactor is in addition coupled to an outflow and to one or more firstpipes or hoses for transferring gases, e.g. environmental air and/or N2gas and/or O2 gas and/or CO2 gas into the bioreactor.

In addition, the bioreactor is coupled to a third pipe or hose for theoff gas. The first pipe or hose may comprise a sensor 144 fordetermining a current total gas influx rate. The third pipe or hose maycomprise a sensor 122, e.g. a CO2 off gas analyzer, to selectivelymeasure the off gas CO2 concentration and the amount of CO2 gastransferred through the third pipe per time unit. According to otherembodiments, there may be not pipes for the influx and outflux ofliquids and nutrients may be fed to the bioreactor by means of step-wisebolus addition of a feed solution.

In many bioreactor types, the influx gasses are fed (as a gas mixture orvia separate openings) into the bioreactor via one or more submersed gasintakes. In case the bioreactor comprises an additional headspaceaeration, the influx rate of said “headspace” influx gas fraction and/orthe air circulation of the gas phase above the medium have to beconfigured such that all gases fed into the bioreactor via headspaceaeration reach pH-CO2 equilibrium with the medium of the bioreactorbefore leaving the bioreactor. Also in case the headspace aeration isthe only aeration mechanism of the bioreactor, the influx rate of said“headspace” influx gas fraction has to be configured such that all gasesfed into the bioreactor reach pH-CO2 equilibrium with the medium of thebioreactor before leaving the bioreactor.

Alternatively (e.g. in case a pH-CO2 equilibrium of the headspaceaeration gases with the medium cannot be reached in time), theadditional headspace aeration is turned off before measuring the CO2concentration in the off gas for performing the pH measuring devicecalibration or offset detection. This may allow avoiding calibrationerrors that could result from a deviation from the equilibrium CO2concentration in the bioreactor gas phase caused by the additionalheadspace aeration.

In case during the initialization phase of the second bioreactor notonly fresh medium but also additional liquids such as feeding solutionsand/or acidic or basic liquids are added to the second bioreactor, thesame amount and composition of said additional liquids is added to thefirst bioreactor during initialization to ensure that at the second andfirst time, the medium (including all the additional liquids andsubstances) in the second and first bioreactors is identical.

FIG. 4 shows diagrams A, B, C and D illustrating the dependence of theCO2 concentration in the off gas of four different bioreactors I-IV fromthe pH value and the independence of said CO2 concentration ofengineering—and size parameters of the respective bioreactors.

The four different bioreactors have the following engineeringproperties:

Bioreactor I Bioreactor II Bioreactor III Bioreactor IV Total volume(volume 0.94 L 1.2 L 1.5 L 1.8 L of medium + gasphase) Aeration rate26.3 mL/L/min 20.8 mL/L/min 16.6 mL/L/min 13.8 mL/L/min Number ofstirrers 1 1 2 2

Each of said bioreactors I-IV was filled with a particular cell culturemedium M1 which did not comprise any cells. The original pH value ofsaid medium was 6.85 (see diagram B). Then, the pH value was increasedin each of the bioreactors by decreasing the CO2 concentration in thegas volume above said medium in the respective bioreactor. At thebeginning of the test and for each of a set of predefined pH values, themedium in each bioreactor was allowed to reach pH-CO2 equilibrium withthe gas volume above the medium at a predefined temperature andpressure, e.g. 20° C. and normal atmospheric pressure. After saidequilibrium was reached, the CO2 concentration in Vol. % of the totaloff gas (also referred to as “fraction CO2 gas”, “CO2[%]” or “FCO2”) wasdetermined for each of said four bioreactors (see diagram A showing, incombination with diagram B, the impact of the pH-value on the measuredCO2 concentration in the off gas). Diagram 4 C) shows the impact of thepH-value on the measured CO2 concentration of each of the fourbioreactors in the form of a bar chart. The maximum deviation of the CO2[%] obtained for each of the four bioreactors was less than 0.4% of thetotal off gas of the bioreactor.

The diagram 4 D) is a plot comprising the CO2 [%] values measured ateach of the four bioreactors I-IV at each of a set of pH values (6.85,6.95, 7.05, 7.15, 7.25, 7.35) at a time when the medium M1 of saidbioreactor reached pH-CO2 equilibrium state.

It should be noted that the pH-CO2 equilibrium in a bioreactor may bechallenged by the rate of CO2 gas entering and/or leaving thebioreactor, so the pH-CO2 equilibrium may in fact be a dynamicequilibrium. Nevertheless, it is possible to control a bioreactor in amanner that the dynamic pH-CO2 equilibrium is established at aparticular pH value, e.g. by decreasing or increasing the CO2concentration in the gas volume above the medium in the bioreactor bymodifying the total CO2 influx rate in the bioreactor. Alternatively,the pH value may be modified by adding acidic or basic substances orliquids.

Preferentially, the dynamic pH-CO2 equilibrium state is established in abioreactor at a particular pH value solely by controlling the CO2 influxrate and total gas outflux rate in a manner that a desired pH value isreached. Using the CO2 concentration for establishing the pH-CO2equilibrium rather than adding a basic or an acidic substance has theadvantage that the composition of the medium is not altered (except forthe concentration of the solved CO2 and its dissociation products) andthus the medium specific relation can be empirically derived from thesame medium at different pH values.

Then, a curve 502 is fitted to the plot in order to empiricallydetermine parameters for a relation 316 being specific for the medium M1contained in the four bioreactors. This approach allows to empiricallydetermine, for a particular cell culture medium, a medium-specificrelation 136 used as input by the comparison unit for predicting theabsolute pH value of a medium given a measured CO2 off gas concentrationwhen said medium has a particular pressure and temperature (e.g. 20° C.and normal atmospheric pressure), lacks any cells and is in pH-CO2equilibrium with the gas phase. The obtained relation is independent ofbioreactor scale, aeration rate and other engineering parameters.

The medium-specific relation is determined only once for a particularmedium M1. The determination may be performed in a single bioreactor,e.g. in the second bioreactor 102 before the second bioreactor isinoculated with the cell culture. In order to increase accuracy, it isalso possible to perform the determination in multiple bioreactors orother containers allowing the measurement of a pH value and a CO2 gasfraction (CO2 concentration) and then use the information obtained inthe multiple bioreactors or containers for obtaining a more accurate,fitted curve 502. In the example depicted in FIGS. 4D and 5 , fourdifferent bioreactors were used for empirically determining a fittedcurve 502 and a corresponding, medium-specific relation betweenequilibrium pH value and equilibrium CO2 concentration.

A further, similar test (not shown) was performed with four bioreactorshaving a volume of 400 L, 100 L, 2 L and 2 L and comprising the sametype of medium. The bioreactors comprised pH measuring devices ofdifferent types (e.g. Knick and Mettler probes), comprised differentcontroller setup configurations (Siemens S7 vs. Sartorius DCU) anddifferent off gas analyzers of the same type (Dasgip/Eppendorf GA4). ThepH measuring devices were calibrated respectively using conventionalcalibration buffers at two known pH points (4 and 7) before they weresubmerged in the medium of their respective bioreactor. In a next step,each of the four pH measuring devices was recalibrated by the use of afifth, pre-calibrated pH measuring device that was sequentially insertedinto the media of the four compared bioreactors. All four pH measuringdevices were recalibrated onto the value of the fifth pH measuringdevice. After that recalibration, the CO₂ concentration in the off gas(“FCO2” value) of all four bioreactors was measured. The four obtainedFCO2 measurement values showed a difference (“delta”) of the maximalvalue of all four values to the minimal value of all four values ofabout 0.75%.

Then, the controller deviation of the four bioreactors was minimized toestablish comparable actual pH values in all four bioreactors. Afterthat minimization, the CO₂ concentration in the off gas (“FCO2” or “CO2[%]) of all four bioreactors was measured. The four obtained FCO2measurement values showed a difference (“delta”) of the maximal value ofall four values to the minimal value of all four values of about 0.27%.The results confirmed that bioreactors, whose media are in pH-CO2equilibrium state have the same CO₂ concentration at the same pH valuesindependent of media volume, overall volume, aeration rate andparameters that depend on aeration rate, stirrer speed and parametersthat depend on stirrer speed and further parameters that depend onscale, bioreactor dimension and the like.

Therefore in said equilibrium state, with a variability of 0.27%, pHoffsets of less than 0.02 pH scale units were detectable in this testscenario. Thus, a highly accurate method for calibrating pH measuringdevices is provided.

FIG. 5 is a transformed version of the diagram in FIG. 4 D). Themedium-specific relation 136 of medium M1 is an equationPPH(pH)=REL-M1(CO2) obtained by mathematically fitting multipleempirically determined pairs of a pH-value and a respective CO2concentration [%] in the gas phase above said medium, the CO2concentration in the gas phase being in pH-CO2 equilibrium with saidmedium according to the Henderson-Hasselbalch equation.

The equation derived empirically by fitting a linear or polynomial curveto the plot of FIG. 5 allows predicting a pH value of a medium at thepredefined temperature and the predefined pressure in case the gasvolume above said medium is in pH-CO2 equilibrium state with said mediumand has a particular CO2 concentration. The prediction is specific forthe medium M1 for which the relation was empirically obtained.

The “CO2” parameter is an input parameter of said equation for inputtinga CO2 concentration measured in a gas phase being in pH-CO2 equilibriumstate with the medium of a bioreactor.

“REL-M1” is a set of one or more parameters a1, a2, b1, b2, b3 connectedby operators. The parameters have been obtained by adjusting samples ofthe medium M1 lacking the cell culture to multiple different pH valuesas described above, thereby letting the samples reach pH-CO2 equilibriumat the predefined pressure and temperature, by determining theequilibrium CO2 concentrations in respective gas volumes being incontact with the medium in the samples, by plotting the measuredequilibrium CO2 concentrations against the respective equilibrium pHvalues of the samples for generating the plot depicted in FIG. 5 ,fitting a curve 502 in the plotted values and deriving the parametersa1, a2 or b1, b2, b3 from the fitted curve.

According to some embodiments, the equation PPH_(M1)(CO2)=REL-M1(CO2) isa linear equation according to PPH_(M1)(CO2)[%]=a1×CO2 [%]+a2. In thiscase, the parameters a1 and a2 are the parameters derived from thefitted curve. In the depicted example, a linear fit would yield thefollowing equation: PPH_(M1)(CO2)=−0.046×CO2[%]+7.45. In this example,a1=−0.046 and a2=7.45.

According to other embodiments, the equation PPH_(M1)(CO2)=REL-M1(CO2)is a polynomial equation according toPPH_(M1)(CO2)=b1×CO2[%]²b2×CO2[%]+b3.

Using a polynomial fit has the advantage that it is more accurate than alinear fit, although a linear fit is already sufficiently accurate forcalculating an absolute pH value by using solely the medium-specificrelation 136 and a CO2 concentration CO2_(R-M-ti) measured e.g. in theoff gas of a bioreactor as input.

FIG. 6 shows the variation of a pH value measured in four differentbioreactors I-IV while growing a cell culture in a particular medium M1over multiple days for a particular cell culture project.Preferentially, each pH value is measured using a pH-measuring device,e.g. a potentiometric pH-meter, immersed in the medium M1 of thebioreactor at pH-CO2 equilibrium of said medium. In each bioreactor, atleast a current pH value and a current CO2 concentration in the off gasare measured repeatedly before and after inoculation and during thewhole project.

For example, the project could be to grow CHO cells (Chinese hamsterovary cells) over 14 days in the cell culture medium M1 under optimal ornearly optimal cell growth conditions until a cell density of about100×10⁵ cells/milliliter is reached.

FIG. 7 shows the CO2 fraction (“CO2 concentration”) measured in the offgas of each of the four bioreactors whose pH value profiles are shown inFIG. 6 . The pH values and CO2 off gas concentrations measured for aparticular bioreactor at a particular moment in time depend on eachother as the CO2 concentration in the gas volume above the mediuminfluences the pH value in accordance with the Henderson-Hasselbalchequation. Moreover, the cell metabolism may have an impact both on thepH value (via excreted metabolites such as lactate) and on the CO2concentration in the gas phase (via aerobic degradation of substrates).

While growing the cells in one of the bioreactors, e.g. in a referencebioreactor 102, the current pH value and the current CO2 off gasconcentrations in the reference bioreactor 102 may be determinedrepeatedly and a derivative parameter value is calculated from at leastsaid two input parameter values and used as a parameter being indicativeof a current status of the cell culture in the reference bioreactor. Aprofile of said derivative parameter values is generated. A profile is arepresentation of the variation of said parameter values versus time.

FIG. 8 a is a diagram showing a reference state profile 402 of areference bioreactor 102, a state profile 802 of a first monitoredand/or controlled bioreactor 104 and a further state profile 804 of afirst monitored/and controlled bioreactor 106. Each state profile isindicative of the state of a bioreactor and its cell culture, wherebythe state at a particular time ti is calculated as a derivative of atleast a currently measured pH value and a currently measured CO2concentration in the off gas of the bioreactor. All bioreactors R, B1and B2 comprise the same medium M1, are operated at the same temperatureand pressure and are in pH-equilibrium state with a respective gasvolume at time t0. The time t0 represents a moment in time just beforethe respective bioreactor is inoculated with a cell culture.

At the moment t0, the reference bioreactor R, also referred to as“second bioreactor”, the first bioreactor B1 and the third bioreactor B2are configured and operated such that they have the same CO2concentration in the off gas. The pH meters of the respectivebioreactors R and B2 may measure an almost identical pH value at timet0. However, the pH measuring device of the bioreactor B1 may measure adifferent pH value at t0 than measured by the pH measuring device of thereference bioreactor (not shown). The pH measuring devices of the threebioreactors may be online pH meters immersed in the medium of therespective bioreactor. In this case, the comparison unit may determinethat there is no calibration difference between the pH meters of thesecond/reference bioreactor R and the pH meter of the first bioreactorB2, but there exists a calibration deviation between the pH meters ofthe reference bioreactor R and the bioreactor B1.

In the depicted example, the profile value of state profile 804 of themonitored bioreactor 106 (“B2”) at time t0 is identical to the referencevalue of the reference profile 402 at time t0. The value of profile 802of the monitored bioreactor 104 (“B4”) at time to significantly differsthe reference value of the reference profile 402 at time t0.

Alternatively, instead of the profile values, the CO2 concentration ofthe off gas of the two bioreactors as depicted in FIG. 7 can be comparedto determine if the pH measuring devices of the two compared bioreactorswere calibrated identically. The two bioreactors are initiated andfilled with the same cell-free medium at the same pressure andtemperature and a current pH value and a current CO2 concentration ofthe medium in the two bioreactors are measured and compared when the twobioreactors have reached pH-CO2 equilibrium. If the CO2 concentration inthe off gas of the two bioreactors are identical while the pH value arenot, or if the pH values of the two bioreactors are identical and theCO2 concentration in the off gas are not, the comparison unit determinesthat the two bioreactors were calibrated differently.

Wrongly calibrated pH measuring devices may result in inaccurate resultswhen comparing the cell culture states of two cell cultures based oncell culture profiles having been derived—solely or in addition to otherparameters—from the pH values. As a consequence, also any action takenby the controller to minimize the state difference may fail to minimizethe state differences (this effect is not shown in FIGS. 8 a and 8 b ,because during the growing of the cell culture in bioreactor B2, thepH-CO2 equilibrium was modified by adding a base and increasing thetotal gas influx rate; thus, the profile of B2 significantly differsfrom the reference profile although the pH meters of the referencebioreactor and of bioreactor B2 were calibrated in the same way.

Wrongly calibrated pH meters may result in inaccurate results whencomparing the cell culture states of two cell cultures based on the pHvalues of the respective bioreactors or any other monitoring or controlparameter being a derivative of said pH values. As a consequence, alsoany action taken by the controller to minimize the pH difference mayfail or may result in an even larger state deviation of the two comparedbioreactors (this effect is not shown in FIGS. 8 a and 8 b , becauseduring the growing of the cell culture in bioreactor B2, the pH-CO2equilibrium was modified by adding a base and increasing the total gasinflux rate; thus, the cell culture state profile of B2 significantlydiffers from the reference state profile although the pH meters of thereference bioreactor and of bioreactor B2 were calibrated in the sameway).

For example, the state profile of a bioreactor before and afterinoculation with a cell culture may be calculated as a PACO profile. APACO value PACO_(B1-ti), PACO_(B2-ti) is indicative of a deviation of aCO2 off gas rate ACO_(B1-M-ti), ACO_(B2-M-ti) measured in the bioreactorfrom a predicted CO2 off gas rate ACO_(B1-EXP-ti), ACO_(B2-EXP-ti). Thepredicted CO2 off gas rate is the off gas rate of said medium in thebioreactor in pH-CO2 equilibrium state under absence of the cell cultureand under the condition that the pH value of the medium in equilibriumstate is identical to the pH value of the bioreactor 104, 106 whenmeasuring the CO2 off gas rate in the bioreactor. The PACO value dependson the amount of CO2 off gas produced by the cells of the cell culturein the bioreactor while cultivating the cell culture. The computation ofthe PACO value PACO_(B1-ti), PACO_(B2-ti) uses as input:

-   -   the received current CO2 off gas rate ACO_(B1-M-ti),        ACO_(B2-M-ti);    -   the received current pH value pH_(B1-ti), pH_(B2-ti);    -   the total gas inflow rate TGI_(B1), TGI_(B2) of the bioreactor        at the time ti of receiving the current CO2-off gas rate; and    -   the medium-specific relation 136.

The computation of the PACO value of the monitored and/or controlledbioreactor at a current time comprises computing, for each of thereceived current CO2 off gas rates and pH values of the monitored and/orcontrolled bioreactor:

-   -   the expected CO2 off gas fraction FCO2_(B1-EXP-ti) of a current        outgas volume of the bioreactor 104 according to:        FCO2_(B1-EXP-ti)=REL-M1(pH_(B1-ti)), wherein FCO2_(B1-EXP-ti) is        a predicted CO2 off gas fraction of the total off gas volume        (TGO_(B1)) of the bioreactor 104 in % at the current time ti,        the prediction being calculated by using the received current pH        value pH_(B1-ti) as input for REL-M1(pH_(B1-ti)), wherein REL-M1        is a medium-specific relation of the medium M1 derived        empirically by fitting a plot such as depicted, for example, in        FIG. 4 D. The parameter pH_(B1-ti) is the received current pH        value in the medium of the bioreactor 104, 106 at a time ti;        thus, the expected CO2 off gas fraction in the bioreactor is        computed under the assumption that the medium of the bioreactor        lacks the cell culture, has the pH value used as input of the        medium-specific relation and is in pH-CO2 equilibrium state with        the gas phase in the bioreactor above said medium and thus is        also in equilibrium with the total off gas volume of said        bioreactor.    -   an expected CO2 off gas rate ACO_(B1-EXP-ti) [mol/min] value        according to:

${{{A{CO}}_{{B1} - {EXP} - {ti}}\lbrack {{mol}/\min} \rbrack} = {( \frac{{F{CO}}2_{{B1} - {EXP} - {{ti}\lbrack\%\rbrack}}}{100} ) \times {TGI}_{B1}}},$wherein the ACO_(B1-EXP-ti) value is the expected CO2 off gas rate ofthe bioreactor (104) when the medium of the bioreactor has the currentlymeasured pH value and is in pH-CO2 equilibrium with the gas phase abovesaid medium, wherein the TGI_(B1) is the total amount of gas influx ofthe bioreactor 104 at the current time (ti); the total amount of gasinflux of the bioreactor is approximately identical to the total amountof gas outflow;

-   -   the PACO_(B1-ti) value according to:        PACO_(B1-ti)=ACO_(B1-EXP-ti)−ACO_(B1-M-ti), wherein        ACO_(B1-M-ti) is the CO2 off gas rate measured at time ti in the        bioreactor 104.

A reference PACO_(B1-ti) value of the reference bioreactor 102 can becomputed accordingly: PACO_(R-ti)=ACO_(R-EXP-ti)−ACO_(R-M-ti), whereinACO_(R-M-ti) is the CO2 off gas rate measured at time ti in thebioreactor 102.

According to some embodiments, the above mentioned comparison of PACOvalues is performed repeatedly after inoculation of the cell culture foridentifying state deviations of the cell culture in bioreactor 104compared to the corresponding cell culture state in the referencebioreactor 102.

A “PACO value” value is a data value. A “FCO2 value” is a data value. An“ACO vale” is a data value. “FCO2” or “CO2 [%]”, also referred to as“CO2 concentration” is the “fraction CO2 gas” in a gas volume, e.g. inthe off gas of a bioreactor.

A “profile” is a set of data values or a mathematical relation thatindicates the variation of a parameter value over time. The parametervalue can be, for example, a PACO value, a CO2 concentration in the offgas (“FCO2”), a CO2 off gas rate (“ACO value”) or the pH value obtainedfrom a bioreactor.

FIG. 8 b is a diagram showing the profile differences of the cellculture state profiles 802, 804 of two bioreactors 104, 106 to thereference profile 402 of the reference bioreactor 102. Curve 810represents profile differences of the bioreactor 104 and the referencebioreactor and curve 808 represents profile differences of thebioreactor 106 and the reference bioreactor. The profile differences ofthe bioreactor 106 to the reference profile 402 are significantly largerthan the differences of the bioreactor 104, because while growing thecell culture in B2, the pH-CO2 equilibrium was modified. Comparing aPACO profile with a reference PACO profile allows identifying cellculture state deviations in two compared bioreactors and toautomatically, semi-automatically or manually take the appropriateactions to minimize profile differences. It has been observed thatcalibration differences between the pH measuring devices may result insignificant differences in control parameter profiles, e.g. PACOprofiles. Thus, using a calibration method according to embodiments ofthe invention in the bioreactor initialization phase may significantlyincrease the accuracy of comparing and synchronizing bioreactor and cellculture states at a later moment in time.

FIG. 9 depicts two box and whisker plots that illustrate that thesampling process has an effect on the measured pH value.

While performing a first cell culture project P1, the pH value of themedium of a bioreactor comprising the cell culture was repeatedlymeasured with a bioreactor-internal pH meter. The pH values measured bythe bioreactor-internal pH meter at multiple time points t1, t2, to werecompared with pH values measured by a second, bioreactor-external pHmeter in medium samples drawn at said respective time points t1, . . . ,tn. Thus, the data values represented by the box and whisker plot ofproject P1 respectively represent the difference between the pH valuemeasured by the bioreactor-internal and the bioreactor-external pH meterat a respective time t1, . . . , tn. Thus, the box and whisker plot forproject P1 depicts the variability and distribution of pH differences(“pH offset effects”) generated by the sampling process. The sampleswere tempered at 32° C. to ensure a constant pH measurement temperaturefor all measurements.

The bioreactor-internal pH meter of project P1 was calibrated accordingto a state of the art method, i.e., by removing the bioreactor-internalpH meter from the bioreactor, calibrating the pH meter outside of thebioreactor with a reference solution of known pH, re-introducing thecalibrated pH meter into the bioreactor and autoclaving the bioreactor.

Moreover, in project P1, the pH values measured by thebioreactor-internal pH meter were repeatedly compared with pH valuesmeasured by the bioreactor-external pH meter in samples of the medium ofthe bioreactor. In case the comparison revealed that a difference (i.e.,“offset”) between the two compared pH values is higher than a giventhreshold, the bioreactor-internal pH meter was recalibrated. Beforeinoculation, recalibration of the bioreactor-internal pH meter tookplace no matter the offset (“focus calibration”). The pH offset ofproject P1 averages around “−0.01” and thus is very close to zero. Thisis not surprising as the pH measurement values obtained by thebioreactor-external pH meter was used as a reference for calibrating thebioreactor-internal pH meter, thereby largely leveling out the offseteffects. A disadvantage of this calibration approach is, however, thatthe absolute, “real” pH value of the medium in the bioreactor and thestrength of the offset effect remains unknown. The variability is veryhigh with only 50% of all data points within +/−0.05 pH, whereas morethan 25% of all offsets are greater than 0.07 pH scale units.

A disparity between on-line and off-line pH measurements (performed bybioreactor-internal and bioreactor-external pH meters) was also observedand confirmed e.g. by Heather Evans et al.: “Dealing with Disparity inOn-line and Off-line pH Measurements Genentech found pH drift in itson-line measurements for a cell culture process, and continues toinvestigate its cause” when performing similar pH measuring and pH metercalibration tests like described for project P1. Heather Evans et al.considered the ability to control the pH within a range of +/−0.10 pHunits as critical for ensuring a consistent and robust processperformance in terms of both productivity and product quality.

The box and whisker plot of the second project P2 was obtained asdescribed for project P1. However, instead of calibrating thebioreactor-internal pH meter according to the state of the art approach,the bioreactor-internal pH meter is calibrated according to anembodiment of the invention using a computed, expected CO2 offgas ratethat was computed for the medium used and for the current temperatureand pressure by taking as input a measured CO2 concentration in the offgas of the bioreactor. Thus, the calibration of the bioreactor-internalpH meter was repeatedly performed (after media fill and theestablishment of a pH-CO2 equilibrium and before inoculation with a cellculture as cell metabolites would shift the equilibrium) using a mediumspecific relation between the pH value and the CO2 off gas rate asdescribed for embodiments of the invention.

The observed pH offset between the extra- and intra-bioreactor pH meteraverages around +0.11, thereby revealing that the strength of the offseteffect is more than 0.1 pH units high. As for P1, the samples were takenat 32° C. and the pH meters used were glass electrodes. The variabilityof offline pH measurement stays comparable, as the sampling procedureand offline pH measurement method in P1 and P2 are the same.

Altogether, 1070 data values were obtained for generating the two boxand whisker plots for projects P1, P2 in FIG. 9 (P1: N=607 and P2:N=463). In both projects, glass electrodes were used at definedtemperature as the intra and extra-bioreactor pH meters.

As can be inferred from the two plots, the variability of the pH offsetsdetermined in both projects P1, P2 is similar. The pH value offsets arecaused by the sampling process in both cases.

However, as can also be inferred from FIG. 9 , the mean of the pHoffsets of project P1 differs from the mean of the offsets obtained forproject P2 by almost 0.1 scale units. This “difference of pH offsetmeans” is caused by different methods used for calibrating thebioreactor-internal pH meters in projects P1 and P2. Differences of themean pH values would also be caused by changing the sampling method,e.g. by increasing the time between taking a sample and actuallyperforming the pH measurement in the sample.

As can be inferred from FIG. 9 , offline pH measurements can be assumedto be the root cause for pH variability. The shift in average offset isdue to general offsets that are added by sampling, sample hold times,temperature drops, carbon dioxide degassing during sampling and offlinemeasurement, as well as specific offsets of the used offline measurementmethod. Blood gas analyzer data (not shown) deliver different offsets.Other offline pH measurement methods (not shown) deliver again differentoffsets.

LIST OF REFERENCE NUMERALS

-   -   100 system for monitoring and/or controlling cell culture states        in a bioreactor    -   102 first (“reference”) bioreactor    -   104 second bioreactor B1    -   106 further bioreactor B2    -   108 pH-measuring device    -   110 processor    -   112 memory    -   114 storage medium    -   120 interface for receiving one or more medium-specific        relations    -   122 CO2 off gas analyzer    -   124 CO2 off gas analyzer    -   126 CO2 off gas analyzer    -   128 interface for receiving measurement parameters from two or        more bioreactors    -   130 comparison unit    -   132 control unit    -   134 display    -   136 medium-specific relation for medium M1    -   138 medium-specific relation for medium M2    -   140 sensor for total gas influx    -   142 pH-measuring device    -   144 sensor for total gas influx    -   146 pH-measuring device    -   202-206 steps    -   402 state profile of reference bioreactor 102    -   502 medium-specific relation plotted for four bioreactors    -   802 state profile of a bioreactor    -   804 state profile of a bioreactor    -   808 state profile difference to reference profile    -   810 state profile difference to reference profile    -   M1 cell culture medium    -   TGI_(B1) total gas influx into bioreactor B1    -   TGI_(B2) total gas influx into bioreactor B2    -   TGI_(R) total gas influx into the reference bioreactor    -   TGO_(B1) total off gas of bioreactor B1    -   TGO_(B2) total off gas of bioreactor B2    -   TGO_(R) total off gas of reference bioreactor    -   TLI_(B1) total liquid influx into bioreactor B1    -   TLI_(B2) total liquid influx into bioreactor B2    -   TLI_(R) total liquid influx into the reference bioreactor    -   TLO_(B1) total (liquid) outflow of bioreactor B1    -   TLO_(B2) total (liquid) outflow of bioreactor B2    -   TLO_(R) total (liquid) outflow of reference bioreactor

The invention claimed is:
 1. A method for determining if a first pHmeasuring device operatively coupled to a first tank is affected by a pHmeasuring problem, the problem being that the first pH measuring deviceis calibrated differently than a second pH measuring device operativelycoupled to a second tank, the method comprising: receiving, by acomparison unit, a first CO2 concentration and a first pH value, thefirst CO2 concentration being a CO2 concentration of a first gas volumeabove a medium in the first tank, the first CO2 concentration and thefirst pH value being measured at a first time, the first time being atime when the medium in the first tank is in pH-CO2 equilibrium statewith the first gas volume at a predefined temperature and pressure, saidequilibrium state being unaffected by the metabolism of any cellculture, the first pH value being a measured value provided by the firstpH measuring device; receiving, by the comparison unit, a second CO2concentration and a second pH value, the second CO2 concentration beinga CO2 concentration of a second gas volume above the same type of mediumcontained in the second tank, the second CO2 concentration and thesecond pH value being measured at a second time, the second time being atime when the medium in the second tank is in pH-CO2 equilibrium statewith the second gas volume at the predefined temperature and pressure,said equilibrium state being unaffected by the metabolism of any cellculture, the second pH value being a measured value provided by thesecond pH measuring device; comparing, by the comparison unit, the firstand second pH values and comparing the first and second CO2concentrations for determining that the first pH measuring device isaffected by the pH measuring problem; calibrating the first pH measuringdevice based on determining that the first pH measuring device isaffected by the pH measuring problem; and measuring a calibrated firstpH value using the calibrated first pH measuring device.
 2. The methodof claim 1, the determination that the first pH measuring device has thepH measuring problem being made in case: the first and second CO2concentrations are identical and the first and second pH values differfrom each other by more than a threshold value; or the first and secondpH values are identical and the first and second CO2 concentrationsdiffer from each other by more than a further threshold value; or afirst data value differs from a second data value by more than a furtherthreshold, the first data value being derived from the first pH valueand the first CO2 concentration, the second data value being derivedfrom the second pH value and the second CO2 concentration.
 3. The methodof claim 1, the first tank being a bioreactor or a harvest tank and/orthe second tank being a reference bioreactor or a reference harvesttank.
 4. The method of claim 1, further comprising: receiving a thirdCO2 concentration and a third pH value, the third CO2 concentrationbeing a CO2 concentration of a third gas volume above the medium in thefirst tank, the third CO2 concentration and the third pH value beingmeasured at a third time, the third time being a time when the medium inthe first tank is in pH-CO2 equilibrium state at the predefinedtemperature and pressure with the third gas volume and after saidequilibrium state is modified by the metabolism of a cell culture in thefirst tank, the third pH value being a measured value provided by thefirst pH measuring device; receiving a fourth CO2 concentration and afourth pH value, the fourth CO2 concentration being a CO2 concentrationof a fourth gas volume above the medium in the second tank, the fourthCO2 concentration and the fourth pH value being measured at a fourthtime, the fourth time being a time when the medium in the second tank isin pH-CO2 equilibrium state at the predefined temperature and pressurewith the second gas volume and after said equilibrium state is modifiedby the metabolism of the cell culture in the second tank, the fourth pHvalue being a measured value provided by the second pH measuring device,a lapsed time between the third time and inoculation of the first tankbeing identical to a lapsed time between the fourth time and inoculationof the second tank; receiving a measured first oxygen uptake rate of thecell culture in the first tank at the third time; receiving a measuredsecond oxygen uptake rate of the cell culture in the second tank at thefourth time; in case the first and second oxygen uptake rates areidentical, comparing the third and fourth pH values and CO2concentrations for determining if the first and second pH measuringdevices are calibrated differently.
 5. The method of claim 1, the firstpH measuring device being at least partially surrounded by the mediumwithin the first tank, wherein: the first tank lacks means for manuallyor automatically taking a sample of the medium in the second tank; orthe first tank comprises means for manually or automatically taking asample of the medium in the first tank, the method further comprising:during a time interval after filling the medium in the first tank andbefore adding a cell culture to the medium in the first tank, keepingall openings of the means for manually or automatically taking a sampleof the medium in the second tank closed.
 6. The method of claim 1, thesecond pH measuring device being at least partially surrounded by themedium within the second tank wherein: the second tank lacks means formanually or automatically taking a sample of the medium in the secondtank; or the second tank comprises means for manually or automaticallytaking a sample of the medium in the second tank, the method furthercomprising: during a time interval after filling the medium in thesecond tank and before adding a cell culture to the medium in the secondtank, keeping all openings of the means for manually or automaticallytaking a sample of the medium in the second tank closed.
 7. The methodof claim 1, the method being used for determining if the second pHmeasuring device is calibrated differently than the first pH measuringdevice, the determination if the first and second pH measuring deviceare calibrated differently being performed while the second pH measuringdevice is at least partially surrounded by the medium in the second tankand without taking a sample of the medium of the second tank forperforming said determination.
 8. The method of claim 1, thedetermination if the first pH measuring device is affected by the pHmeasuring problem being performed by using the first and second CO2concentrations and the first and second pH values as the only data inputfor said determination.
 9. The method of claim 1, the method furthercomprising: performing an online-measurement with the first pH measuringdevice for measuring the first pH value, the first pH measuring devicebeing at least partially surrounded with the medium in the first tank;and/or performing an online-measurement by a first CO2 sensor in the offgas of the first tank for providing the first CO2 concentration.
 10. Themethod of claim 1, further comprising: performing an online-measurementwith the second pH measuring device for measuring the second pH value,the second pH measuring device being at least partially surrounded withthe medium in the second tank; and/or performing an online-measurementby a second CO2 sensor in the off gas of the second tank for providingthe second CO2 concentration.
 11. The method of claim 1, the methodcomprising, in case of determining that the first pH measuring device isaffected by the pH measuring problem, performing one or more of thefollowing steps by the comparison unit: outputting a warning message;automatically performing or triggering the performing of the calibratingof the first pH measuring device; automatically performing or triggeringthe performing of a replacement of the first pH measuring device by anew first pH measuring device.
 12. The method of claim 1, the first tankdiffering from the second tank in respect to one or more of thefollowing features: a) the gas volume in the tank, b) the medium volumein the tank, c) the Reynolds number of the tank, d) the Newton number ofthe tank, e) the dimensions of the tank, f) geometrical features of thetank and/or tank baffles, g) the stirrer configuration, h) the stirringrate, i) the volumetric mass transfer coefficient for oxygen (kLa) ofthe tank, j) total gas influx rate and/or O2 influx rate and/or N2influx rate and/or CO2 influx rate, k) power input, l) pressure in thetank, m)gas bubble hold time in the medium, n) gas bubble size anddistribution in the medium, o) surface speed, p) a parameter calculatedas a derivative from one or more of the parameters a)-o); q) thegeographic location of the two tanks.
 13. A system comprising: a firsttank, a first pH measuring device, a second tank, and a second pHmeasuring device, and a comparison unit, the system configured for:receiving, by the comparison unit, a first CO2 concentration and a firstpH value, the first CO2 concentration being a CO2 concentration of afirst gas volume above a medium in the first tank, the first CO2concentration and the first pH value being measured at a first time, thefirst time being a time when the medium in the first tank is in pH-CO2equilibrium state with the first gas volume at a predefined temperatureand pressure, said equilibrium state being unaffected by the metabolismof any cell culture, the first pH value being a measured value providedby the first pH measuring device; receiving, by the comparison unit, asecond CO2 concentration and a second pH value, the second CO2concentration being a CO2 concentration of a second gas volume above thesame type of medium contained in the second tank, the second CO2concentration and the second pH value being measured at a second time,the second time being a time when the medium in the second tank is inpH-CO2 equilibrium state with the second gas volume at the predefinedtemperature and pressure, said equilibrium state being unaffected by themetabolism of any cell culture, the second pH value being a measuredvalue provided by the second pH measuring device; comparing the firstand second pH values and comparing the first and second CO2concentrations for determining that the first pH measuring device isaffected by a pH measuring problem, the problem being that the first pHmeasuring device operatively coupled to the first tank is calibrateddifferently than the second pH measuring device operatively coupled tothe second tank; calibrating the first pH measuring device based ondetermining that the first pH measuring device is affected by the pHmeasuring problem; and measuring a calibrated first pH value using thecalibrated first pH measuring device.
 14. The system of claim 13,further comprising: a control unit operatively coupled to the comparisonunit; the control unit being configured to monitor and/or control astate of a cell culture in the first tank, thereby using pH valuesrepeatedly measured by the first pH measuring device as input.
 15. Thesystem of claim 13, wherein the determination that the first pHmeasuring device has a pH measuring problem is made in case: the firstand second CO2 concentrations are identical and the first and second pHvalues differ from each other by more than a threshold value; or thefirst and second pH values are identical and the first and second CO2concentrations differ from each other by more than a further thresholdvalue; or a first data value differs from a second data value by morethan a further threshold, the first data value being derived from thefirst pH value and the first CO2 concentration, the second data valuebeing derived from the second pH value and the second CO2 concentration.16. The system of claim 13, the first tank being a bioreactor or aharvest tank and/or the second tank being a reference bioreactor or areference harvest tank.
 17. The system of claim 13, further configuredto: receive a third CO2 concentration and a third pH value, the thirdCO2 concentration being a CO2 concentration of a third gas volume abovethe medium in the first tank, the third CO2 concentration and the thirdpH value being measured at a third time, the third time being a timewhen the medium in the first tank is in pH-CO2 equilibrium state at thepredefined temperature and pressure with the third gas volume and aftersaid equilibrium state is modified by the metabolism of a cell culturein the first tank, the third pH value being a measured value provided bythe first pH measuring device; receive a fourth CO2 concentration and afourth pH value, the fourth CO2 concentration being a CO2 concentrationof a fourth gas volume above the medium in the second tank, the fourthCO2 concentration and the fourth pH value being measured at a fourthtime, the fourth time being a time when the medium in the second tank isin pH-CO2 equilibrium state at the predefined temperature and pressurewith the second gas volume and after said equilibrium state is modifiedby the metabolism of the cell culture in the second tank, the fourth pHvalue being a measured value provided by the second pH measuring device,a lapsed time between the third time and inoculation of the first tankbeing identical to a lapsed time between the fourth time and inoculationof the second tank; receive a measured first oxygen uptake rate of thecell culture in the first tank at the third time; receive a measuredsecond oxygen uptake rate of the cell culture in the second tank at thefourth time; in case the first and second oxygen uptake rates areidentical, compare the third and fourth pH values and CO2 concentrationsfor determining if the first and second pH measuring devices arecalibrated differently.
 18. The system of claim 13, the first pHmeasuring device being at least partially surrounded by the mediumwithin the first tank, wherein: the first tank lacks means for manuallyor automatically taking a sample of the medium in the second tank; orthe first tank comprises means for manually or automatically taking asample of the medium in the first tank, the method further comprising:during a time interval after filling the medium in the first tank andbefore adding a cell culture to the medium in the first tank, keepingall openings of the means for manually or automatically taking a sampleof the medium in the second tank closed.
 19. The system of claim 13, thesecond pH measuring device being at least partially surrounded by themedium within the second tank wherein: the second tank lacks means formanually or automatically taking a sample of the medium in the secondtank; or the second tank comprises means for manually or automaticallytaking a sample of the medium in the second tank, the method furthercomprising: during a time interval after filling the medium in thesecond tank and before adding a cell culture to the medium in the secondtank, keeping all openings of the means for manually or automaticallytaking a sample of the medium in the second tank closed.
 20. At leastone non-transitory computer-readable storage medium comprising computerreadable instructions that, when executed by at least one processor, areconfigured to cause the at least one processor to: receive a first CO2concentration and a first pH value, the first CO2 concentration being aCO2 concentration of a first gas volume above a medium in a first tank,the first CO2 concentration and the first pH value being measured at afirst time, the first time being a time when the medium in the firsttank is in pH-CO2 equilibrium state with the first gas volume at apredefined temperature and pressure, said equilibrium state beingunaffected by the metabolism of any cell culture, the first pH valuebeing a measured value provided by a first pH measuring device; receivea second CO2 concentration and a second pH value, the second CO2concentration being a CO2 concentration of a second gas volume above thesame type of medium contained in a second tank, the second CO2concentration and the second pH value being measured at a second time,the second time being a time when the medium in the second tank is inpH-CO2 equilibrium state with the second gas volume at the predefinedtemperature and pressure, said equilibrium state being unaffected by themetabolism of any cell culture, the second pH value being a measuredvalue provided by a second pH measuring device; compare the first andsecond pH values and comparing compare the first and second CO2concentrations for determining that the first pH measuring device isaffected by a pH measuring problem; calibrate the first pH measuringdevice based on determining that the first pH measuring device isaffected by the pH measuring problem, the problem being that the firstpH measuring device operatively coupled to the first tank is calibrateddifferently than the second pH measuring device operatively coupled tothe second tank; and cause measurement of a calibrated first pH valueusing the calibrated first pH measuring device.